Introduction

The largest challenge facing the usage of metagenomic approaches in microbiology is the need to extend traditional microbiology training to include metagenomic or sequencing data analysis. Sean Eddy (a compuational biologist at the Howard Hughes Medical Institute) nicely describes the impacts of high throughput sequencing on biology and its training in his keynote address (http://cryptogenomicon.org/2014/11/01/high-throughput-sequencing-for-neuroscience/#more-858).

To facilitate the barriers to microbiologists for metagenomic assembly, we have complemented this review with a tutorial of how to estimate the abundance of reference sequences (e.g., genes, contigs, etc.) in a metagenome. We include approaches that include using references that are both (i) available genome references or (ii) assembled from the metagenome. In general, to complete this tutorial and most metagenomic assembly, one would need:

  • Access to a server. Most metagenomic assembly will require more memory than most researchers will have on their personal computers. In this tutorial, we will provide training on the publicly accessible Amazon EC2 instances which can be rented by anyone with a registered account.
  • Access to a metagenomic dataset. We have selected the usage of the HMP Mock Community WGS dataset (http://www.hmpdacc.org/HMMC/) for this tutorial given its availability, practical size, and availability of reference genomes. This dataset represents a mock metagenome of 22 known organisms for which DNA was extracted from cultured isolates, combined, and sequenced.
  • Software for assembly, read mapping, and annotation. We will demonstrate the installation of this software on an Ubuntu-based server.

0. Getting on the same page.

The first step in this tutorial is to provide all users access to a server for which these instructions can be used, regardless of what computer you may be on. To do this, we'll be using cloud computing. More specifically, Amazon Web Services Elastic Compute Cloud. To rent compute time off of Amazon Web Services, you'll have to sign up and pay with a credit card. The cost is pretty manageable, http://aws.amazon.com/ec2/pricing/. You should be able to complete this tutorial in less than four hours, which comes out to < $1.

Once you are signed up for Amazon Web Services, you need to follow some instructions to launch a cloud "instance" or server. For this tutorial, we suggest you use the instructions from the Data Science Toolbox, http://datasciencetoolbox.org/#bundles. A couple things to note prior to running through those instructions:

  • Choose the "in the cloud" instructions
  • You can choose any AMI but we suggest US EAST, ami-d1737bb8
  • When you configure your instance, choose the m3.large instance
  • Do not forget to "Add rule" as described in the directions in Data Science Toolbox Step 2: Add a "custom TCP rule" for port "8888" and source "Anywhere".
  • Complete the Data Science Instructions through Step 4. Once you get to Step 5, refer below.

If you have trouble logging into your instance and you are on a Mac or Linux OS:

  • Check to see that you changed permissions on your key file (the one that ends in *.pem)
  • Make sure you run the ssh command to log into the instance in the same directory as your security file or specify the location of that file

Once you are logged into the instance, for example, you've successfully run the following command (except you'll have your own security file uniquely named and your own special EC2 address):

$ ssh -i MyKeyPair.pem ubuntu@ec2-XX-XX-XX-XXX.compute-1.amazonaws.com

And you now have a command line that looks like:

ubuntu@ip-10-181-106-120:

You need to do a couple things to get this tutorial running:

Copy and paste the following commands one by one into your command line and press ENTER after each one:

cd /mnt

sudo git clone https://github.com/germs-lab/frontiers-review-2015.git


Next, copy and paste the following command and enter a notebook password of your choice when prompted:

dst setup base

Then, copy and paste this command:

sudo ipython notebook --profile=dst --notebook-dir=/mnt/frontiers-review-2015

This will start up an IPython Notebook for this tutorial. Leave the terminal screen open and find your internet browser, preferablly Google Chrome. You'll also need the address for your EC2 instance public DNS that you used to log in above "e.g., ec2-XX-XX-XX-XXX". If you don't know it, you can always check on your AWS EC2 dashboard (see running instances) here, https://console.aws.amazon.com/ec2/v2/.

On your web browser, navigate to https://ec2-XX-XX-XX-XXX:8888 (except with your specific EC2 public DNS). Almost all web browsers will have a message that says you're heading to an unsafe place. Don't be alarmed. On Chrome, you can hit the "Advanced" options link and hit "Proceed anyways". Then, type in the password (password is the one you chose above), and voila, you'll see a notebook that contains this text called "frontiers-nb-2015".

1. How to use this IPython Notebook

IPython notebooks are very useful to collaboratively train bioinformatics. These notebooks have recently been featured in Nature News (http://www.nature.com/news/interactive-notebooks-sharing-the-code-1.16261 and http://www.nature.com/news/programming-pick-up-python-1.16833)

In using these notebooks, there are a few imporant things to note. There are two types of content in this tutorial: text and code. This content is placed in this notebook as "cells". If you click around on this page, you'll see different cells highlighted. To execute each cell (regardless of content), you hit on your keyboard SHIFT+ENTER. If the cell contains text, the content will be displayed directly. If the cell contains code, the code will then execute. Also, you can execute all cells in the notebook by going to the Cell tab where "File, Edit, View, Insert, Cell..." are in the top left of this webpage and selecting Run all.

2. Download the tutorial dataset.

We will begin this tutorial by downloading the HMP mock metagenome from the NCBI Short Read Archives (SRA). Many public metagenomes are stored as SRA files in the NCBI. The easiest way to get these SRA files is to use a special set of tools called the sratoolkit. If you have your dataset SRA run ID (in this case SRR172903), you can download the dataset and convert it to the standard "fasta" or "fastq" sequencing format is to use a special program to convert the file.


In [1]:
!wget http://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/2.4.5-2/sratoolkit.2.4.5-2-ubuntu64.tar.gz


--2015-06-26 13:48:38--  http://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/2.4.5-2/sratoolkit.2.4.5-2-ubuntu64.tar.gz
Resolving ftp-trace.ncbi.nlm.nih.gov (ftp-trace.ncbi.nlm.nih.gov)... 130.14.250.11, 2607:f220:41e:250::10
Connecting to ftp-trace.ncbi.nlm.nih.gov (ftp-trace.ncbi.nlm.nih.gov)|130.14.250.11|:80... connected.
HTTP request sent, awaiting response... 200 OK
Length: 62432226 (60M) [application/x-gzip]
Saving to: ‘sratoolkit.2.4.5-2-ubuntu64.tar.gz’

100%[======================================>] 62,432,226  73.7MB/s   in 0.8s   

2015-06-26 13:48:39 (73.7 MB/s) - ‘sratoolkit.2.4.5-2-ubuntu64.tar.gz’ saved [62432226/62432226]


In [2]:
!tar -xvf sratoolkit.2.4.5-2-ubuntu64.tar.gz


sratoolkit.2.4.5-2-ubuntu64/
sratoolkit.2.4.5-2-ubuntu64/README
sratoolkit.2.4.5-2-ubuntu64/README-blastn
sratoolkit.2.4.5-2-ubuntu64/README-vdb-config
sratoolkit.2.4.5-2-ubuntu64/bin/
sratoolkit.2.4.5-2-ubuntu64/bin/ncbi/
sratoolkit.2.4.5-2-ubuntu64/bin/ncbi/default.kfg
sratoolkit.2.4.5-2-ubuntu64/bin/ncbi/vdb-copy.kfg
sratoolkit.2.4.5-2-ubuntu64/bin/abi-dump
sratoolkit.2.4.5-2-ubuntu64/bin/abi-dump.2
sratoolkit.2.4.5-2-ubuntu64/bin/abi-dump.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/abi-load
sratoolkit.2.4.5-2-ubuntu64/bin/abi-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/abi-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/align-info
sratoolkit.2.4.5-2-ubuntu64/bin/align-info.2
sratoolkit.2.4.5-2-ubuntu64/bin/align-info.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/bam-load
sratoolkit.2.4.5-2-ubuntu64/bin/bam-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/bam-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/blastn_vdb
sratoolkit.2.4.5-2-ubuntu64/bin/blastn_vdb.2
sratoolkit.2.4.5-2-ubuntu64/bin/blastn_vdb.2.2.30-2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/cache-mgr
sratoolkit.2.4.5-2-ubuntu64/bin/cache-mgr.2
sratoolkit.2.4.5-2-ubuntu64/bin/cache-mgr.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/cg-load
sratoolkit.2.4.5-2-ubuntu64/bin/cg-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/cg-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/fastq-dump
sratoolkit.2.4.5-2-ubuntu64/bin/fastq-dump.2
sratoolkit.2.4.5-2-ubuntu64/bin/fastq-dump.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/fastq-load
sratoolkit.2.4.5-2-ubuntu64/bin/fastq-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/fastq-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/helicos-load
sratoolkit.2.4.5-2-ubuntu64/bin/helicos-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/helicos-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/illumina-dump
sratoolkit.2.4.5-2-ubuntu64/bin/illumina-dump.2
sratoolkit.2.4.5-2-ubuntu64/bin/illumina-dump.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/illumina-load
sratoolkit.2.4.5-2-ubuntu64/bin/illumina-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/illumina-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/kar
sratoolkit.2.4.5-2-ubuntu64/bin/kar.2
sratoolkit.2.4.5-2-ubuntu64/bin/kar.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/kdbmeta
sratoolkit.2.4.5-2-ubuntu64/bin/kdbmeta.2
sratoolkit.2.4.5-2-ubuntu64/bin/kdbmeta.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/latf-load
sratoolkit.2.4.5-2-ubuntu64/bin/latf-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/latf-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/pacbio-load
sratoolkit.2.4.5-2-ubuntu64/bin/pacbio-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/pacbio-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/prefetch
sratoolkit.2.4.5-2-ubuntu64/bin/prefetch.2
sratoolkit.2.4.5-2-ubuntu64/bin/prefetch.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/rcexplain
sratoolkit.2.4.5-2-ubuntu64/bin/rcexplain.2
sratoolkit.2.4.5-2-ubuntu64/bin/rcexplain.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/remote-fuser
sratoolkit.2.4.5-2-ubuntu64/bin/remote-fuser.2
sratoolkit.2.4.5-2-ubuntu64/bin/remote-fuser.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/sam-dump
sratoolkit.2.4.5-2-ubuntu64/bin/sam-dump.2
sratoolkit.2.4.5-2-ubuntu64/bin/sam-dump.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/sff-dump
sratoolkit.2.4.5-2-ubuntu64/bin/sff-dump.2
sratoolkit.2.4.5-2-ubuntu64/bin/sff-dump.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/sff-load
sratoolkit.2.4.5-2-ubuntu64/bin/sff-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/sff-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/sra-kar
sratoolkit.2.4.5-2-ubuntu64/bin/sra-kar.2
sratoolkit.2.4.5-2-ubuntu64/bin/sra-kar.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/sra-pileup
sratoolkit.2.4.5-2-ubuntu64/bin/sra-pileup.2
sratoolkit.2.4.5-2-ubuntu64/bin/sra-pileup.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/sra-sort
sratoolkit.2.4.5-2-ubuntu64/bin/sra-sort.2
sratoolkit.2.4.5-2-ubuntu64/bin/sra-sort.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/sra-stat
sratoolkit.2.4.5-2-ubuntu64/bin/sra-stat.2
sratoolkit.2.4.5-2-ubuntu64/bin/sra-stat.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/srapath
sratoolkit.2.4.5-2-ubuntu64/bin/srapath.2
sratoolkit.2.4.5-2-ubuntu64/bin/srapath.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/srf-load
sratoolkit.2.4.5-2-ubuntu64/bin/srf-load.2
sratoolkit.2.4.5-2-ubuntu64/bin/srf-load.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/tblastn_vdb
sratoolkit.2.4.5-2-ubuntu64/bin/tblastn_vdb.2
sratoolkit.2.4.5-2-ubuntu64/bin/tblastn_vdb.2.2.30-2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/test-sra
sratoolkit.2.4.5-2-ubuntu64/bin/test-sra.2
sratoolkit.2.4.5-2-ubuntu64/bin/test-sra.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-config
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-config.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-config.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-copy
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-copy.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-copy.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-decrypt
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-decrypt.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-decrypt.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-dump
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-dump.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-dump.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-encrypt
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-encrypt.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-encrypt.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-lock
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-lock.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-lock.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-passwd
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-passwd.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-passwd.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-unlock
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-unlock.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-unlock.2.4.5
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-validate
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-validate.2
sratoolkit.2.4.5-2-ubuntu64/bin/vdb-validate.2.4.5
sratoolkit.2.4.5-2-ubuntu64/example/
sratoolkit.2.4.5-2-ubuntu64/example/perl/
sratoolkit.2.4.5-2-ubuntu64/example/perl/base-stats.pl
sratoolkit.2.4.5-2-ubuntu64/example/perl/dump-reference.pl
sratoolkit.2.4.5-2-ubuntu64/example/perl/gene-lookup.pl
sratoolkit.2.4.5-2-ubuntu64/example/perl/mismatch-stats.pl
sratoolkit.2.4.5-2-ubuntu64/example/perl/quality-stats.pl
sratoolkit.2.4.5-2-ubuntu64/example/perl/simplefastq.pl
sratoolkit.2.4.5-2-ubuntu64/example/perl/splitfastq.pl
sratoolkit.2.4.5-2-ubuntu64/schema/
sratoolkit.2.4.5-2-ubuntu64/schema/align/
sratoolkit.2.4.5-2-ubuntu64/schema/align/align.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/align/mate-cache.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/align/qstat.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/align/refseq.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/align/seq.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/insdc/
sratoolkit.2.4.5-2-ubuntu64/schema/insdc/insdc.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/insdc/seq.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/insdc/sra.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/clip.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/ncbi.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/pnbrdb.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/seq-graph.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/seq.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/spotname.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/sra.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/stats.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/varloc.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/ncbi/wgs-contig.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/sra/
sratoolkit.2.4.5-2-ubuntu64/schema/sra/454.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/sra/abi.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/sra/helicos.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/sra/illumina.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/sra/ion-torrent.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/sra/pacbio.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/sra/pevents.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/vdb/
sratoolkit.2.4.5-2-ubuntu64/schema/vdb/built-in.vschema
sratoolkit.2.4.5-2-ubuntu64/schema/vdb/vdb.vschema

You can see that we now have a file containing the software with the "ls" command. You'll also see this notebook in the list of files in the present location we are working in.


In [3]:
!ls


assemstats3.py		    khmer-install.sh
bowtie.sh		    kmer-count-plot.py
fastx_install.sh	    LICENSE.md
fetch-genomes-fasta.py	    ncbi_acc.txt
fraggenescan-install.sh     README.md
frontiers-nb-2015.bu.ipynb  remove_output.py
frontiers-nb-2015.ipynb     sratoolkit.2.4.5-2-ubuntu64
install-megahit.sh	    sratoolkit.2.4.5-2-ubuntu64.tar.gz
ipython_notebook_config.py  unique-kmers.py
khmer

Now we'll use the installed sratoolkit program to download the HMP mock dataset in "fastq" format. (This takes a minute or two. You'll find that patience is require for working with metagenomes. The nice thing about working in the cloud is that you are "renting" the computational power so it is not using your personal computer's memory -- freeing it up for things you can do while waiting. You'll note that you can see that "Kernel busy" will be shown on the top-right corner of the screen below "Logout" button.)


In [4]:
!sratoolkit.2.4.5-2-ubuntu64/bin/fastq-dump SRR172903


Read 7932819 spots for SRR172903
Written 7932819 spots for SRR172903

3. Quality control

There are a number of methods to determine the quality of the sequencing data that you will assemble. First, one can look at the quality scores of your sequencing reads and if desired, trim reads with quality scores that are not sufficient for your needs. A vast number of tools are available to perform quality trimming of sequencing reads, including tools with nice tutorials including FastX Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/ and http://khmer-protocols.readthedocs.org/en/v0.8-1/metagenomics/1-quality.html), FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ and http://ged.msu.edu/angus/tutorials-2013/short-read-quality-evaluation.html) and Sickle (https://github.com/najoshi/sickle and http://2014-5-metagenomics-workshop.readthedocs.org/en/latest/assembly/qtrim.html).

The sequencing data file you have downloaded is a "fastq" text file, where data describing each sequencing read is shown on four lines. Let's take a quick look:


In [5]:
!head -n 4 SRR172903.fastq


@SRR172903.1 USI-EAS376:2:1:2:1414 length=75
CTTACCACCAGGAACNAACTTTGAGATTCCAAAAGATCGAGTACCAGAGGGATGGACAGTAACAGTAGATCCAGA
+SRR172903.1 USI-EAS376:2:1:2:1414 length=75
BB@BBBABB????<=%04@@>A?6><<AB@7:@A?@>A@AA8@>59==???887905<?6>25=###########
  • This first line (starting with "@SRR172903.1") is the read identifier, it usually shows the read ID, some information for the sequencing facility about the run that it was obtained on.
  • The second line is the DNA sequence.
  • The third line is the same as the first line but replacing the "@" with a "+", sometimes this is only a "+" in some datasets
  • The fourth line gives you information on the quality score of each base pair for the DNA sequence. Note that it is the same length as the DNA sequence and that quality scores are based on ASCII character scores (with an offset determined by the sequencing technology, Illumina is currently an offset of 64, e.g., ASCII code 64 = 0 Phred score). The quality score is equal to the -10 * log (p), where p is the probability of the base being called wrong (e.g., if Q= 20, p=0.01, 1% probability base is called wrong).

For this tutorial, we will be removing reads that have more than 50% of the read length with a Phred score of less than 33. We will be using Fastx-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/commandline.html) which can be used for many types of quality control (e.g., adapter trimming). First, we will download, uncompress, and then install this program.


In [6]:
!wget https://github.com/agordon/fastx_toolkit/releases/download/0.0.14/fastx_toolkit-0.0.14.tar.bz2
!wget https://github.com/agordon/libgtextutils/releases/download/0.7/libgtextutils-0.7.tar.gz


--2015-06-26 13:49:56--  https://github.com/agordon/fastx_toolkit/releases/download/0.0.14/fastx_toolkit-0.0.14.tar.bz2
Resolving github.com (github.com)... 192.30.252.131
Connecting to github.com (github.com)|192.30.252.131|:443... connected.
HTTP request sent, awaiting response... 302 Found
Location: https://s3.amazonaws.com/github-cloud/releases/14233196/f0821c9e-764e-11e3-99f0-a5603899c05f.bz2?response-content-disposition=attachment%3B%20filename%3Dfastx_toolkit-0.0.14.tar.bz2&response-content-type=application/octet-stream&AWSAccessKeyId=AKIAISTNZFOVBIJMK3TQ&Expires=1435326656&Signature=Djr691B6%2B06IT0vVIfSLO%2FT1PF8%3D [following]
--2015-06-26 13:49:56--  https://s3.amazonaws.com/github-cloud/releases/14233196/f0821c9e-764e-11e3-99f0-a5603899c05f.bz2?response-content-disposition=attachment%3B%20filename%3Dfastx_toolkit-0.0.14.tar.bz2&response-content-type=application/octet-stream&AWSAccessKeyId=AKIAISTNZFOVBIJMK3TQ&Expires=1435326656&Signature=Djr691B6%2B06IT0vVIfSLO%2FT1PF8%3D
Resolving s3.amazonaws.com (s3.amazonaws.com)... 54.231.13.104
Connecting to s3.amazonaws.com (s3.amazonaws.com)|54.231.13.104|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 543018 (530K) [application/octet-stream]
Saving to: ‘fastx_toolkit-0.0.14.tar.bz2’

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In [7]:
!tar -xvf fastx_toolkit-0.0.14.tar.bz2
!tar -xvf libgtextutils-0.7.tar.gz


fastx_toolkit-0.0.14/
fastx_toolkit-0.0.14/Makefile.in
fastx_toolkit-0.0.14/m4/
fastx_toolkit-0.0.14/m4/Makefile.in
fastx_toolkit-0.0.14/m4/ax_cxx_header_stdcxx_tr1.m4
fastx_toolkit-0.0.14/m4/libtool.m4
fastx_toolkit-0.0.14/m4/ax_c_long_long.m4
fastx_toolkit-0.0.14/m4/Makefile.am
fastx_toolkit-0.0.14/m4/ax_cxx_compile_stdcxx_11.m4
fastx_toolkit-0.0.14/m4/lt~obsolete.m4
fastx_toolkit-0.0.14/m4/ltsugar.m4
fastx_toolkit-0.0.14/m4/ltversion.m4
fastx_toolkit-0.0.14/m4/ltoptions.m4
fastx_toolkit-0.0.14/doc/
fastx_toolkit-0.0.14/doc/Makefile.in
fastx_toolkit-0.0.14/doc/Makefile.am
fastx_toolkit-0.0.14/config.h.in
fastx_toolkit-0.0.14/Makefile.am
fastx_toolkit-0.0.14/install_galaxy_files.sh
fastx_toolkit-0.0.14/src/
fastx_toolkit-0.0.14/src/Makefile.in
fastx_toolkit-0.0.14/src/fastq_quality_converter/
fastx_toolkit-0.0.14/src/fastq_quality_converter/Makefile.in
fastx_toolkit-0.0.14/src/fastq_quality_converter/fastq_quality_converter.c
fastx_toolkit-0.0.14/src/fastq_quality_converter/Makefile.am
fastx_toolkit-0.0.14/src/fastq_quality_filter/
fastx_toolkit-0.0.14/src/fastq_quality_filter/Makefile.in
fastx_toolkit-0.0.14/src/fastq_quality_filter/Makefile.am
fastx_toolkit-0.0.14/src/fastq_quality_filter/fastq_quality_filter.c
fastx_toolkit-0.0.14/src/libfastx/
fastx_toolkit-0.0.14/src/libfastx/Makefile.in
fastx_toolkit-0.0.14/src/libfastx/fastx_args.c
fastx_toolkit-0.0.14/src/libfastx/Makefile.am
fastx_toolkit-0.0.14/src/libfastx/sequence_alignment.h
fastx_toolkit-0.0.14/src/libfastx/fastx_args.h
fastx_toolkit-0.0.14/src/libfastx/fastx.h
fastx_toolkit-0.0.14/src/libfastx/fastx.c
fastx_toolkit-0.0.14/src/libfastx/sequence_alignment.cpp
fastx_toolkit-0.0.14/src/libfastx/chomp.h
fastx_toolkit-0.0.14/src/libfastx/chomp.c
fastx_toolkit-0.0.14/src/fastx_trimmer/
fastx_toolkit-0.0.14/src/fastx_trimmer/Makefile.in
fastx_toolkit-0.0.14/src/fastx_trimmer/Makefile.am
fastx_toolkit-0.0.14/src/fastx_trimmer/fastx_trimmer.c
fastx_toolkit-0.0.14/src/Makefile.am
fastx_toolkit-0.0.14/src/fastx_reverse_complement/
fastx_toolkit-0.0.14/src/fastx_reverse_complement/Makefile.in
fastx_toolkit-0.0.14/src/fastx_reverse_complement/Makefile.am
fastx_toolkit-0.0.14/src/fastx_reverse_complement/fastx_reverse_complement.c
fastx_toolkit-0.0.14/src/fastx_collapser/
fastx_toolkit-0.0.14/src/fastx_collapser/Makefile.in
fastx_toolkit-0.0.14/src/fastx_collapser/fastx_collapser.cpp
fastx_toolkit-0.0.14/src/fastx_collapser/Makefile.am
fastx_toolkit-0.0.14/src/fastx_collapser/std_hash.h
fastx_toolkit-0.0.14/src/fasta_nucleotide_changer/
fastx_toolkit-0.0.14/src/fasta_nucleotide_changer/Makefile.in
fastx_toolkit-0.0.14/src/fasta_nucleotide_changer/fasta_nucleotide_changer.c
fastx_toolkit-0.0.14/src/fasta_nucleotide_changer/Makefile.am
fastx_toolkit-0.0.14/src/fastx_uncollapser/
fastx_toolkit-0.0.14/src/fastx_uncollapser/Makefile.in
fastx_toolkit-0.0.14/src/fastx_uncollapser/Makefile.am
fastx_toolkit-0.0.14/src/fastx_uncollapser/fastx_uncollapser.cpp
fastx_toolkit-0.0.14/src/fastq_quality_trimmer/
fastx_toolkit-0.0.14/src/fastq_quality_trimmer/Makefile.in
fastx_toolkit-0.0.14/src/fastq_quality_trimmer/Makefile.am
fastx_toolkit-0.0.14/src/fastq_quality_trimmer/fastq_quality_trimmer.c
fastx_toolkit-0.0.14/src/fastq_to_fasta/
fastx_toolkit-0.0.14/src/fastq_to_fasta/Makefile.in
fastx_toolkit-0.0.14/src/fastq_to_fasta/Makefile.am
fastx_toolkit-0.0.14/src/fastq_to_fasta/fastq_to_fasta.c
fastx_toolkit-0.0.14/src/fastx_renamer/
fastx_toolkit-0.0.14/src/fastx_renamer/Makefile.in
fastx_toolkit-0.0.14/src/fastx_renamer/fastx_renamer.c
fastx_toolkit-0.0.14/src/fastx_renamer/Makefile.am
fastx_toolkit-0.0.14/src/fastx_artifacts_filter/
fastx_toolkit-0.0.14/src/fastx_artifacts_filter/Makefile.in
fastx_toolkit-0.0.14/src/fastx_artifacts_filter/Makefile.am
fastx_toolkit-0.0.14/src/fastx_artifacts_filter/fastx_artifacts_filter.c
fastx_toolkit-0.0.14/src/fastq_masker/
fastx_toolkit-0.0.14/src/fastq_masker/Makefile.in
fastx_toolkit-0.0.14/src/fastq_masker/Makefile.am
fastx_toolkit-0.0.14/src/fastq_masker/fastq_masker.c
fastx_toolkit-0.0.14/src/fastx_quality_stats/
fastx_toolkit-0.0.14/src/fastx_quality_stats/Makefile.in
fastx_toolkit-0.0.14/src/fastx_quality_stats/Makefile.am
fastx_toolkit-0.0.14/src/fastx_quality_stats/fastx_quality_stats.c
fastx_toolkit-0.0.14/src/seqalign_test/
fastx_toolkit-0.0.14/src/seqalign_test/Makefile.in
fastx_toolkit-0.0.14/src/seqalign_test/Makefile.am
fastx_toolkit-0.0.14/src/seqalign_test/seqalign_test.cpp
fastx_toolkit-0.0.14/src/fastx_clipper/
fastx_toolkit-0.0.14/src/fastx_clipper/Makefile.in
fastx_toolkit-0.0.14/src/fastx_clipper/fastx_clipper.cpp
fastx_toolkit-0.0.14/src/fastx_clipper/Makefile.am
fastx_toolkit-0.0.14/src/fasta_formatter/
fastx_toolkit-0.0.14/src/fasta_formatter/Makefile.in
fastx_toolkit-0.0.14/src/fasta_formatter/Makefile.am
fastx_toolkit-0.0.14/src/fasta_formatter/sequence_writers.h
fastx_toolkit-0.0.14/src/fasta_formatter/fasta_formatter.cpp
fastx_toolkit-0.0.14/NEWS
fastx_toolkit-0.0.14/README
fastx_toolkit-0.0.14/configure.ac
fastx_toolkit-0.0.14/ChangeLog
fastx_toolkit-0.0.14/scripts/
fastx_toolkit-0.0.14/scripts/Makefile.in
fastx_toolkit-0.0.14/scripts/fastx_barcode_splitter.pl
fastx_toolkit-0.0.14/scripts/fastx_nucleotide_distribution_line_graph.sh
fastx_toolkit-0.0.14/scripts/fastx_nucleotide_distribution_graph.sh
fastx_toolkit-0.0.14/scripts/Makefile.am
fastx_toolkit-0.0.14/scripts/fasta_clipping_histogram.pl
fastx_toolkit-0.0.14/scripts/fastq_quality_boxplot_graph.sh
fastx_toolkit-0.0.14/build_scripts/
fastx_toolkit-0.0.14/build_scripts/Makefile.in
fastx_toolkit-0.0.14/build_scripts/build_on_opensolaris_2009.6.sh
fastx_toolkit-0.0.14/build_scripts/Makefile.am
fastx_toolkit-0.0.14/build_scripts/build_on_linux.sh
fastx_toolkit-0.0.14/COPYING
fastx_toolkit-0.0.14/galaxy/
fastx_toolkit-0.0.14/galaxy/Makefile.in
fastx_toolkit-0.0.14/galaxy/static/
fastx_toolkit-0.0.14/galaxy/static/Makefile.in
fastx_toolkit-0.0.14/galaxy/static/Makefile.am
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/Makefile.in
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastq_quality_boxplot_1.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastq_nucleotides_distribution_4.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/barcode_splitter_output_example.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastq_quality_boxplot_2.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fasta_clipping_histogram_1.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/Makefile.am
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fasta_clipping_histogram_3.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastq_nucleotides_distribution_2.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastq_nucleotides_distribution_line_graph.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fasta_clipping_histogram_4.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastx_clipper_illustration.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastq_nucleotides_distribution_1.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastx_clipper_example.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fasta_clipping_histogram_2.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastq_quality_boxplot_3.png
fastx_toolkit-0.0.14/galaxy/static/fastx_icons/fastq_nucleotides_distribution_3.png
fastx_toolkit-0.0.14/galaxy/test-data/
fastx_toolkit-0.0.14/galaxy/test-data/fastx_rev_comp2.fastq
fastx_toolkit-0.0.14/galaxy/test-data/Makefile.in
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_conv1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_trimmer1.fasta
fastx_toolkit-0.0.14/galaxy/test-data/fastx_trimmer2.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_barcode_splitter1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastq_stats1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastq_stats1.fastq
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_filter1a.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_artifacts2.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_reverse_complement1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_conv2n.out
fastx_toolkit-0.0.14/galaxy/test-data/fasta_collapser1.fasta
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_conv2.fastq
fastx_toolkit-0.0.14/galaxy/test-data/fasta_collapser1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastq_to_fasta1b.out
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_filter1.fastq
fastx_toolkit-0.0.14/galaxy/test-data/fastx_trimmer2.fastq
fastx_toolkit-0.0.14/galaxy/test-data/Makefile.am
fastx_toolkit-0.0.14/galaxy/test-data/fasta_nuc_changer1.fasta
fastx_toolkit-0.0.14/galaxy/test-data/fasta_nuc_changer2.fasta
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_filter1b.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_artifacts1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_conv2.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_artifacts2.fastq
fastx_toolkit-0.0.14/galaxy/test-data/fastq_to_fasta1.fastq
fastx_toolkit-0.0.14/galaxy/test-data/fastx_barcode_splitter1.txt
fastx_toolkit-0.0.14/galaxy/test-data/fastx_trimmer1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_reverse_complement2.out
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_conv1.fastq
fastx_toolkit-0.0.14/galaxy/test-data/fastq_to_fasta1a.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_artifacts1.fasta
fastx_toolkit-0.0.14/galaxy/test-data/fastq_qual_conv1a.out
fastx_toolkit-0.0.14/galaxy/test-data/fasta_nuc_changer1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_clipper1a.out
fastx_toolkit-0.0.14/galaxy/test-data/fasta_nuc_changer2.out
fastx_toolkit-0.0.14/galaxy/test-data/fasta_formatter2.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_rev_comp1.fasta
fastx_toolkit-0.0.14/galaxy/test-data/fasta_formatter1.out
fastx_toolkit-0.0.14/galaxy/test-data/fastx_clipper1.fastq
fastx_toolkit-0.0.14/galaxy/test-data/fasta_formatter1.fasta
fastx_toolkit-0.0.14/galaxy/test-data/fastx_barcode_splitter1.fastq
fastx_toolkit-0.0.14/galaxy/Makefile.am
fastx_toolkit-0.0.14/galaxy/README
fastx_toolkit-0.0.14/galaxy/tools/
fastx_toolkit-0.0.14/galaxy/tools/Makefile.in
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/Makefile.in
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_barcode_splitter.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_barcode_splitter_galaxy_wrapper.sh
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_trimmer.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_clipper.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fasta_clipping_histogram.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_nucleotides_distribution_line.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/Makefile.am
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastq_to_fasta.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_nucleotides_distribution.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_reverse_complement.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastq_masker.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_uncollapser.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_artifacts_filter.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_quality_statistics.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_renamer.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastq_quality_trimmer.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_trimmer_from_end.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/seqid_uncollapser.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastq_quality_filter.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastq_quality_boxplot.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastx_collapser.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fasta_formatter.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fastq_quality_converter.xml
fastx_toolkit-0.0.14/galaxy/tools/fastx_toolkit/fasta_nucleotide_changer.xml
fastx_toolkit-0.0.14/galaxy/tools/Makefile.am
fastx_toolkit-0.0.14/galaxy/tool-data/
fastx_toolkit-0.0.14/galaxy/tool-data/Makefile.in
fastx_toolkit-0.0.14/galaxy/tool-data/fastx_clipper_sequences.txt
fastx_toolkit-0.0.14/galaxy/tool-data/Makefile.am
fastx_toolkit-0.0.14/galaxy/fastx_toolkit_conf.xml
fastx_toolkit-0.0.14/INSTALL
fastx_toolkit-0.0.14/aclocal.m4
fastx_toolkit-0.0.14/AUTHORS
fastx_toolkit-0.0.14/reconf
fastx_toolkit-0.0.14/THANKS
fastx_toolkit-0.0.14/config/
fastx_toolkit-0.0.14/config/install-sh
fastx_toolkit-0.0.14/config/compile
fastx_toolkit-0.0.14/config/missing
fastx_toolkit-0.0.14/config/depcomp
fastx_toolkit-0.0.14/config/config.guess
fastx_toolkit-0.0.14/config/ltmain.sh
fastx_toolkit-0.0.14/config/config.sub
fastx_toolkit-0.0.14/configure
libgtextutils-0.7/
libgtextutils-0.7/Makefile.in
libgtextutils-0.7/m4/
libgtextutils-0.7/m4/Makefile.in
libgtextutils-0.7/m4/libtool.m4
libgtextutils-0.7/m4/Makefile.am
libgtextutils-0.7/m4/lt~obsolete.m4
libgtextutils-0.7/m4/ltsugar.m4
libgtextutils-0.7/m4/ltversion.m4
libgtextutils-0.7/m4/ltoptions.m4
libgtextutils-0.7/doc/
libgtextutils-0.7/doc/Makefile.in
libgtextutils-0.7/doc/Makefile.am
libgtextutils-0.7/gtextutils.pc.in
libgtextutils-0.7/config.h.in
libgtextutils-0.7/Makefile.am
libgtextutils-0.7/src/
libgtextutils-0.7/src/Makefile.in
libgtextutils-0.7/src/gtextutils/
libgtextutils-0.7/src/gtextutils/Makefile.in
libgtextutils-0.7/src/gtextutils/outbuf3.hpp
libgtextutils-0.7/src/gtextutils/text_line_reader.h
libgtextutils-0.7/src/gtextutils/pipe_fitter.c
libgtextutils-0.7/src/gtextutils/stream_wrapper.cpp
libgtextutils-0.7/src/gtextutils/tuple_parser.h
libgtextutils-0.7/src/gtextutils/Makefile.am
libgtextutils-0.7/src/gtextutils/string_tokenize.h
libgtextutils-0.7/src/gtextutils/pipe_fitter.h
libgtextutils-0.7/src/gtextutils/stream_wrapper.h
libgtextutils-0.7/src/gtextutils/strnatcmp.h
libgtextutils-0.7/src/gtextutils/exit_manip.h
libgtextutils-0.7/src/gtextutils/inbuf1.hpp
libgtextutils-0.7/src/gtextutils/text_line_reader.cpp
libgtextutils-0.7/src/gtextutils/strnatcmp.c
libgtextutils-0.7/src/gtextutils/container_join.h
libgtextutils-0.7/src/gtextutils/natsort.h
libgtextutils-0.7/src/Makefile.am
libgtextutils-0.7/NEWS
libgtextutils-0.7/README
libgtextutils-0.7/configure.ac
libgtextutils-0.7/ChangeLog
libgtextutils-0.7/COPYING
libgtextutils-0.7/INSTALL
libgtextutils-0.7/aclocal.m4
libgtextutils-0.7/tests/
libgtextutils-0.7/tests/test_string_tokenize.cpp
libgtextutils-0.7/tests/Makefile.in
libgtextutils-0.7/tests/test_tuple_parser.cpp
libgtextutils-0.7/tests/test_tuple_parser_file.cpp
libgtextutils-0.7/tests/test_fd_inbuf.cpp
libgtextutils-0.7/tests/test_text_reader_unget.cpp
libgtextutils-0.7/tests/test_container_join.cpp
libgtextutils-0.7/tests/Makefile.am
libgtextutils-0.7/tests/test_pipe_fitter.c
libgtextutils-0.7/tests/test_natural_sort.cpp
libgtextutils-0.7/tests/tests_assertion.h
libgtextutils-0.7/tests/test_text_reader.cpp
libgtextutils-0.7/tests/test_in_out_buf.cpp
libgtextutils-0.7/tests/test_input_stream_wrapper.cpp
libgtextutils-0.7/tests/test_fd_outbuf.cpp
libgtextutils-0.7/AUTHORS
libgtextutils-0.7/reconf
libgtextutils-0.7/THANKS
libgtextutils-0.7/config/
libgtextutils-0.7/config/install-sh
libgtextutils-0.7/config/compile
libgtextutils-0.7/config/missing
libgtextutils-0.7/config/test-driver
libgtextutils-0.7/config/depcomp
libgtextutils-0.7/config/config.guess
libgtextutils-0.7/config/ltmain.sh
libgtextutils-0.7/config/config.sub
libgtextutils-0.7/configure

In [16]:
!bash fastx_install.sh




g++ is already the newest version.
gcc is already the newest version.
pkg-config is already the newest version.
0 upgraded, 0 newly installed, 0 to remove and 14 not upgraded.
checking for a BSD-compatible install... /usr/bin/install -c
checking whether build environment is sane... yes
/mnt/frontiers-review-2015/libgtextutils-0.7/config/missing: Unknown `--is-lightweight' option
Try `/mnt/frontiers-review-2015/libgtextutils-0.7/config/missing --help' for more information
configure: WARNING: 'missing' script is too old or missing
checking for a thread-safe mkdir -p... /bin/mkdir -p
checking for gawk... gawk
checking whether make sets $(MAKE)... yes
checking whether make supports nested variables... yes
checking for gcc... gcc
checking whether the C compiler works... yes
checking for C compiler default output file name... a.out
checking for suffix of executables... 
checking whether we are cross compiling... no
checking for suffix of object files... o
checking whether we are using the GNU C compiler... yes
checking whether gcc accepts -g... yes
checking for gcc option to accept ISO C89... none needed
checking whether gcc understands -c and -o together... yes
checking for style of include used by make... GNU
checking dependency style of gcc... gcc3
checking for g++... g++
checking whether we are using the GNU C++ compiler... yes
checking whether g++ accepts -g... yes
checking dependency style of g++... gcc3
checking build system type... x86_64-unknown-linux-gnu
checking host system type... x86_64-unknown-linux-gnu
checking for a sed that does not truncate output... /bin/sed
checking for grep that handles long lines and -e... /bin/grep
checking for egrep... /bin/grep -E
checking for fgrep... /bin/grep -F
checking for ld used by gcc... /usr/bin/ld
checking if the linker (/usr/bin/ld) is GNU ld... yes
checking for BSD- or MS-compatible name lister (nm)... /usr/bin/nm -B
checking the name lister (/usr/bin/nm -B) interface... BSD nm
checking whether ln -s works... yes
checking the maximum length of command line arguments... 1572864
checking whether the shell understands some XSI constructs... yes
checking whether the shell understands "+="... yes
checking for /usr/bin/ld option to reload object files... -r
checking for objdump... objdump
checking how to recognize dependent libraries... pass_all
checking for ar... ar
checking for strip... strip
checking for ranlib... ranlib
checking command to parse /usr/bin/nm -B output from gcc object... ok
checking how to run the C preprocessor... gcc -E
checking for ANSI C header files... yes
checking for sys/types.h... yes
checking for sys/stat.h... yes
checking for stdlib.h... yes
checking for string.h... yes
checking for memory.h... yes
checking for strings.h... yes
checking for inttypes.h... yes
checking for stdint.h... yes
checking for unistd.h... yes
checking for dlfcn.h... yes
checking whether we are using the GNU C++ compiler... (cached) yes
checking whether g++ accepts -g... (cached) yes
checking dependency style of g++... (cached) gcc3
checking how to run the C++ preprocessor... g++ -E
checking for objdir... .libs
checking if gcc supports -fno-rtti -fno-exceptions... no
checking for gcc option to produce PIC... -fPIC -DPIC
checking if gcc PIC flag -fPIC -DPIC works... yes
checking if gcc static flag -static works... yes
checking if gcc supports -c -o file.o... yes
checking if gcc supports -c -o file.o... (cached) yes
checking whether the gcc linker (/usr/bin/ld -m elf_x86_64) supports shared libraries... yes
checking whether -lc should be explicitly linked in... no
checking dynamic linker characteristics... GNU/Linux ld.so
checking how to hardcode library paths into programs... immediate
checking whether stripping libraries is possible... yes
checking if libtool supports shared libraries... yes
checking whether to build shared libraries... yes
checking whether to build static libraries... yes
checking for ld used by g++... /usr/bin/ld -m elf_x86_64
checking if the linker (/usr/bin/ld -m elf_x86_64) is GNU ld... yes
checking whether the g++ linker (/usr/bin/ld -m elf_x86_64) supports shared libraries... yes
checking for g++ option to produce PIC... -fPIC -DPIC
checking if g++ PIC flag -fPIC -DPIC works... yes
checking if g++ static flag -static works... yes
checking if g++ supports -c -o file.o... yes
checking if g++ supports -c -o file.o... (cached) yes
checking whether the g++ linker (/usr/bin/ld -m elf_x86_64) supports shared libraries... yes
checking dynamic linker characteristics... GNU/Linux ld.so
checking how to hardcode library paths into programs... immediate
checking that generated files are newer than configure... done
configure: creating ./config.status
config.status: creating Makefile
config.status: creating doc/Makefile
config.status: creating m4/Makefile
config.status: creating src/Makefile
config.status: creating src/gtextutils/Makefile
config.status: creating gtextutils.pc
config.status: creating tests/Makefile
config.status: creating config.h
config.status: config.h is unchanged
config.status: executing depfiles commands
config.status: executing libtool commands
make  all-recursive
make[1]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7'
Making all in m4
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/m4'
make[2]: Nothing to be done for `all'.
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/m4'
Making all in src
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
Making all in gtextutils
make[3]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src/gtextutils'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src/gtextutils'
make[3]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
make[3]: Nothing to be done for `all-am'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
Making all in doc
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/doc'
make[2]: Nothing to be done for `all'.
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/doc'
Making all in tests
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/tests'
make[2]: Nothing to be done for `all'.
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/tests'
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7'
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7'
make[1]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7'
Making install in m4
make[1]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/m4'
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/m4'
make[2]: Nothing to be done for `install-exec-am'.
 /bin/mkdir -p '/usr/local/share/aclocal'
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/m4'
make[1]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/m4'
Making install in src
make[1]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
Making install in gtextutils
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src/gtextutils'
make[3]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src/gtextutils'
 /bin/mkdir -p '/usr/local/lib'
 /bin/bash ../../libtool   --mode=install /usr/bin/install -c   libgtextutils.la '/usr/local/lib'
libtool: install: /usr/bin/install -c .libs/libgtextutils-0.7.so.0.0.0 /usr/local/lib/libgtextutils-0.7.so.0.0.0
libtool: install: (cd /usr/local/lib && { ln -s -f libgtextutils-0.7.so.0.0.0 libgtextutils-0.7.so.0 || { rm -f libgtextutils-0.7.so.0 && ln -s libgtextutils-0.7.so.0.0.0 libgtextutils-0.7.so.0; }; })
libtool: install: (cd /usr/local/lib && { ln -s -f libgtextutils-0.7.so.0.0.0 libgtextutils.so || { rm -f libgtextutils.so && ln -s libgtextutils-0.7.so.0.0.0 libgtextutils.so; }; })
libtool: install: /usr/bin/install -c .libs/libgtextutils.lai /usr/local/lib/libgtextutils.la
libtool: install: /usr/bin/install -c .libs/libgtextutils.a /usr/local/lib/libgtextutils.a
libtool: install: chmod 644 /usr/local/lib/libgtextutils.a
libtool: install: ranlib /usr/local/lib/libgtextutils.a
libtool: finish: PATH="/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin:/sbin:/bin:/sbin" ldconfig -n /usr/local/lib
----------------------------------------------------------------------
Libraries have been installed in:
   /usr/local/lib

If you ever happen to want to link against installed libraries
in a given directory, LIBDIR, you must either use libtool, and
specify the full pathname of the library, or use the `-LLIBDIR'
flag during linking and do at least one of the following:
   - add LIBDIR to the `LD_LIBRARY_PATH' environment variable
     during execution
   - add LIBDIR to the `LD_RUN_PATH' environment variable
     during linking
   - use the `-Wl,-rpath -Wl,LIBDIR' linker flag
   - have your system administrator add LIBDIR to `/etc/ld.so.conf'

See any operating system documentation about shared libraries for
more information, such as the ld(1) and ld.so(8) manual pages.
----------------------------------------------------------------------
 /bin/mkdir -p '/usr/local/include/gtextutils/gtextutils'
 /usr/bin/install -c -m 644 container_join.h text_line_reader.h stream_wrapper.h natsort.h strnatcmp.h outbuf3.hpp inbuf1.hpp tuple_parser.h exit_manip.h string_tokenize.h pipe_fitter.h '/usr/local/include/gtextutils/gtextutils'
make[3]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src/gtextutils'
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src/gtextutils'
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
make[3]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
make[3]: Nothing to be done for `install-exec-am'.
make[3]: Nothing to be done for `install-data-am'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
make[1]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/src'
Making install in doc
make[1]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/doc'
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/doc'
make[2]: Nothing to be done for `install-exec-am'.
make[2]: Nothing to be done for `install-data-am'.
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/doc'
make[1]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/doc'
Making install in tests
make[1]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/tests'
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7/tests'
make[2]: Nothing to be done for `install-exec-am'.
make[2]: Nothing to be done for `install-data-am'.
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/tests'
make[1]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7/tests'
make[1]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7'
make[2]: Entering directory `/mnt/frontiers-review-2015/libgtextutils-0.7'
make[2]: Nothing to be done for `install-exec-am'.
 /bin/mkdir -p '/usr/local/lib/pkgconfig'
 /usr/bin/install -c -m 644 gtextutils.pc '/usr/local/lib/pkgconfig'
make[2]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7'
make[1]: Leaving directory `/mnt/frontiers-review-2015/libgtextutils-0.7'
checking for a BSD-compatible install... /usr/bin/install -c
checking whether build environment is sane... yes
checking for a thread-safe mkdir -p... /bin/mkdir -p
checking for gawk... gawk
checking whether make sets $(MAKE)... yes
checking whether make supports nested variables... yes
checking for gcc... gcc
checking whether the C compiler works... yes
checking for C compiler default output file name... a.out
checking for suffix of executables... 
checking whether we are cross compiling... no
checking for suffix of object files... o
checking whether we are using the GNU C compiler... yes
checking whether gcc accepts -g... yes
checking for gcc option to accept ISO C89... none needed
checking whether gcc understands -c and -o together... yes
checking for style of include used by make... GNU
checking dependency style of gcc... gcc3
checking for g++... g++
checking whether we are using the GNU C++ compiler... yes
checking whether g++ accepts -g... yes
checking dependency style of g++... gcc3
checking build system type... x86_64-unknown-linux-gnu
checking host system type... x86_64-unknown-linux-gnu
checking for a sed that does not truncate output... /bin/sed
checking for grep that handles long lines and -e... /bin/grep
checking for egrep... /bin/grep -E
checking for fgrep... /bin/grep -F
checking for ld used by gcc... /usr/bin/ld
checking if the linker (/usr/bin/ld) is GNU ld... yes
checking for BSD- or MS-compatible name lister (nm)... /usr/bin/nm -B
checking the name lister (/usr/bin/nm -B) interface... BSD nm
checking whether ln -s works... yes
checking the maximum length of command line arguments... 1572864
checking whether the shell understands some XSI constructs... yes
checking whether the shell understands "+="... yes
checking for /usr/bin/ld option to reload object files... -r
checking for objdump... objdump
checking how to recognize dependent libraries... pass_all
checking for ar... ar
checking for strip... strip
checking for ranlib... ranlib
checking command to parse /usr/bin/nm -B output from gcc object... ok
checking how to run the C preprocessor... gcc -E
checking for ANSI C header files... yes
checking for sys/types.h... yes
checking for sys/stat.h... yes
checking for stdlib.h... yes
checking for string.h... yes
checking for memory.h... yes
checking for strings.h... yes
checking for inttypes.h... yes
checking for stdint.h... yes
checking for unistd.h... yes
checking for dlfcn.h... yes
checking whether we are using the GNU C++ compiler... (cached) yes
checking whether g++ accepts -g... (cached) yes
checking dependency style of g++... (cached) gcc3
checking how to run the C++ preprocessor... g++ -E
checking for objdir... .libs
checking if gcc supports -fno-rtti -fno-exceptions... no
checking for gcc option to produce PIC... -fPIC -DPIC
checking if gcc PIC flag -fPIC -DPIC works... yes
checking if gcc static flag -static works... yes
checking if gcc supports -c -o file.o... yes
checking if gcc supports -c -o file.o... (cached) yes
checking whether the gcc linker (/usr/bin/ld -m elf_x86_64) supports shared libraries... yes
checking whether -lc should be explicitly linked in... no
checking dynamic linker characteristics... GNU/Linux ld.so
checking how to hardcode library paths into programs... immediate
checking whether stripping libraries is possible... yes
checking if libtool supports shared libraries... yes
checking whether to build shared libraries... yes
checking whether to build static libraries... yes
checking for ld used by g++... /usr/bin/ld -m elf_x86_64
checking if the linker (/usr/bin/ld -m elf_x86_64) is GNU ld... yes
checking whether the g++ linker (/usr/bin/ld -m elf_x86_64) supports shared libraries... yes
checking for g++ option to produce PIC... -fPIC -DPIC
checking if g++ PIC flag -fPIC -DPIC works... yes
checking if g++ static flag -static works... yes
checking if g++ supports -c -o file.o... yes
checking if g++ supports -c -o file.o... (cached) yes
checking whether the g++ linker (/usr/bin/ld -m elf_x86_64) supports shared libraries... yes
checking dynamic linker characteristics... GNU/Linux ld.so
checking how to hardcode library paths into programs... immediate
checking for long long int... yes
checking for ISO C++ TR1 include files... yes
checking whether g++ supports C++11 features by default... no
checking whether g++ supports C++11 features with -std=c++11... yes
checking for pkg-config... /usr/bin/pkg-config
checking pkg-config is at least version 0.9.0... yes
checking for GTEXTUTILS... yes
checking that generated files are newer than configure... done
configure: creating ./config.status
config.status: creating Makefile
config.status: creating doc/Makefile
config.status: creating m4/Makefile
config.status: creating src/Makefile
config.status: creating src/libfastx/Makefile
config.status: creating src/fastx_clipper/Makefile
config.status: creating src/fastq_to_fasta/Makefile
config.status: creating src/fastx_quality_stats/Makefile
config.status: creating src/fastq_quality_converter/Makefile
config.status: creating src/fastx_trimmer/Makefile
config.status: creating src/fastq_quality_filter/Makefile
config.status: creating src/fastq_quality_trimmer/Makefile
config.status: creating src/fastx_artifacts_filter/Makefile
config.status: creating src/fastx_reverse_complement/Makefile
config.status: creating src/fastx_collapser/Makefile
config.status: creating src/fastx_uncollapser/Makefile
config.status: creating src/seqalign_test/Makefile
config.status: creating src/fasta_formatter/Makefile
config.status: creating src/fasta_nucleotide_changer/Makefile
config.status: creating src/fastx_renamer/Makefile
config.status: creating src/fastq_masker/Makefile
config.status: creating galaxy/Makefile
config.status: creating galaxy/tools/Makefile
config.status: creating galaxy/tools/fastx_toolkit/Makefile
config.status: creating galaxy/test-data/Makefile
config.status: creating galaxy/static/Makefile
config.status: creating galaxy/static/fastx_icons/Makefile
config.status: creating galaxy/tool-data/Makefile
config.status: creating scripts/Makefile
config.status: creating build_scripts/Makefile
config.status: creating config.h
config.status: config.h is unchanged
config.status: executing depfiles commands
config.status: executing libtool commands
make  all-recursive
make[1]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14'
Making all in m4
make[2]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/m4'
make[2]: Nothing to be done for `all'.
make[2]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/m4'
Making all in src
make[2]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src'
Making all in libfastx
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/libfastx'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/libfastx'
Making all in fastx_clipper
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_clipper'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_clipper'
Making all in fastx_trimmer
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_trimmer'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_trimmer'
Making all in fastx_quality_stats
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_quality_stats'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_quality_stats'
Making all in fastq_quality_converter
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastq_quality_converter'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastq_quality_converter'
Making all in fastq_to_fasta
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastq_to_fasta'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastq_to_fasta'
Making all in fastq_quality_filter
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastq_quality_filter'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastq_quality_filter'
Making all in fastq_quality_trimmer
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastq_quality_trimmer'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastq_quality_trimmer'
Making all in fastx_artifacts_filter
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_artifacts_filter'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_artifacts_filter'
Making all in fastx_reverse_complement
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_reverse_complement'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_reverse_complement'
Making all in fastx_collapser
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_collapser'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_collapser'
Making all in fastx_uncollapser
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_uncollapser'
g++ -DHAVE_CONFIG_H -I. -I../..   -I/usr/local/include/gtextutils   -I../../src/libfastx   -g -O2 -std=c++11 -Wall -Wextra -Wformat-nonliteral -Wformat-security -Wswitch-default -Wswitch-enum -Wunused-parameter -Wfloat-equal -Werror -DDEBUG -g -O1 -MT fastx_uncollapser.o -MD -MP -MF .deps/fastx_uncollapser.Tpo -c -o fastx_uncollapser.o fastx_uncollapser.cpp
mv -f .deps/fastx_uncollapser.Tpo .deps/fastx_uncollapser.Po
/bin/bash ../../libtool  --tag=CXX   --mode=link g++  -g -O2 -std=c++11 -Wall -Wextra -Wformat-nonliteral -Wformat-security -Wswitch-default -Wswitch-enum -Wunused-parameter -Wfloat-equal -Werror -DDEBUG -g -O1   -o fastx_uncollapser fastx_uncollapser.o -L/usr/local/lib -lgtextutils   ../libfastx/libfastx.a  
libtool: link: g++ -g -O2 -std=c++11 -Wall -Wextra -Wformat-nonliteral -Wformat-security -Wswitch-default -Wswitch-enum -Wunused-parameter -Wfloat-equal -Werror -DDEBUG -g -O1 -o fastx_uncollapser fastx_uncollapser.o  -L/usr/local/lib /usr/local/lib/libgtextutils.so ../libfastx/libfastx.a
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fastx_uncollapser'
Making all in seqalign_test
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/seqalign_test'
make[3]: Nothing to be done for `all'.
make[3]: Leaving directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/seqalign_test'
Making all in fasta_formatter
make[3]: Entering directory `/mnt/frontiers-review-2015/fastx_toolkit-0.0.14/src/fasta_formatter'
g++ -DHAVE_CONFIG_H -I. -I../..   -I/usr/local/include/gtextutils   -I../../src/libfastx   -g -O2 -std=c++11 -Wall -Wextra -Wformat-nonliteral -Wformat-security -Wswitch-default -Wswitch-enum -Wunused-parameter -Wfloat-equal -Werror -DDEBUG -g -O1 -MT fasta_formatter.o -MD -MP -MF .deps/fasta_formatter.Tpo -c -o fasta_formatter.o fasta_formatter.cpp
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 /bin/mkdir -p '/usr/local/bin'
 /usr/bin/install -c fastx_barcode_splitter.pl fastx_nucleotide_distribution_graph.sh fastx_nucleotide_distribution_line_graph.sh fastq_quality_boxplot_graph.sh fasta_clipping_histogram.pl '/usr/local/bin'
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Now, we will perform the quality filtering saving the quality-filtered file as SRR172903.qc.fastq:

FASTQ Quality Filter

$ fastq_quality_filter -h usage: fastq_quality_filter [-h] [-v] [-q N] [-p N] [-z] [-i INFILE] [-o OUTFILE]

version 0.0.6
   [-h]         = This helpful help screen.
   [-q N]       = Minimum quality score to keep.
   [-p N]       = Minimum percent of bases that must have [-q] quality.
   [-z]         = Compress output with GZIP.
   [-i INFILE]  = FASTA/Q input file. default is STDIN.
   [-o OUTFILE] = FASTA/Q output file. default is STDOUT.
   [-v]         = Verbose - report number of sequences.
          If [-o] is specified,  report will be printed to STDOUT.
          If [-o] is not specified (and output goes to STDOUT),
          report will be printed to STDERR.

In [17]:
!fastq_quality_filter -q 33 -p 50 -i SRR172903.fastq > SRR172903.qc.fastq

4. Checking the diversity - What is the distribution of "Who is There?"

An advantage of metagenomic sequencing is the ability to quantify microbial diversity in an environment without the need to first cultivate cells. Typically, most studies access taxonomic diversity (especially with the usage of targeted sequencing of the 16S rRNA gene*). Diversity can also be measured in the representation of specific sequence patterns in a metagenome. For example, one can quantify the abundance of unique nucleotide "words" of length K, or k-mers, in a metagenome. These k-mers can also be used in the assembly of metagenomes where overlapping k-mers are indicative of reads that should be connected together. The diversity of these k-mers can give you insight into the the diversity of your sample. Further, since assembly compares each k-mer against all k-mers, larger numbers of k-mers present will require more computational memory. A nice review on k-mers and assembly is Miller et al.

(*Note that 16S rRNA amplicon sequencing is a targeted approach and not considered metagenomics in this review. Shotgun metagenomic sequencing uses DNA extracted from all cells in a community and sequenced. Targeted sequencing amplifies a specific genomic locus and independently sequenced. A great review on metagenome analysis is Sharpton et al.)

The first thing we will do is install khmer (www.github.com/ged-lab/khmer) -- it contains a suite of khmer and pre-assembly tools. We will use it for k-mer counting here. Once you run the script below, you can use khmer's many tools.


In [18]:
!ls


assemstats3.py		      kmer-count-plot.py
bowtie.sh		      libgtextutils-0.7
fastx_install.sh	      libgtextutils-0.7.tar.gz
fastx_toolkit-0.0.14	      LICENSE.md
fastx_toolkit-0.0.14.tar.bz2  ncbi_acc.txt
fetch-genomes-fasta.py	      README.md
fraggenescan-install.sh       remove_output.py
frontiers-nb-2015.bu.ipynb    sratoolkit.2.4.5-2-ubuntu64
frontiers-nb-2015.ipynb       sratoolkit.2.4.5-2-ubuntu64.tar.gz
install-megahit.sh	      SRR172903.fastq
ipython_notebook_config.py    SRR172903.qc.fastq
khmer			      unique-kmers.py
khmer-install.sh

In [19]:
!bash khmer-install.sh


Cloning into 'khmer'...
remote: Counting objects: 33672, done.
remote: Compressing objects: 100% (153/153), done.
remote: Total 33672 (delta 82), reused 0 (delta 0), pack-reused 33519
Receiving objects: 100% (33672/33672), 107.68 MiB | 4.25 MiB/s, done.
Resolving deltas: 100% (22993/22993), done.
Checking connectivity... done
running install
Checking .pth file support in /usr/local/lib/python2.7/dist-packages/
/usr/bin/python -E -c pass
TEST PASSED: /usr/local/lib/python2.7/dist-packages/ appears to support .pth files
running bdist_egg
running egg_info
creating khmer.egg-info
writing requirements to khmer.egg-info/requires.txt
writing khmer.egg-info/PKG-INFO
writing top-level names to khmer.egg-info/top_level.txt
writing dependency_links to khmer.egg-info/dependency_links.txt
writing entry points to khmer.egg-info/entry_points.txt
writing manifest file 'khmer.egg-info/SOURCES.txt'
reading manifest file 'khmer.egg-info/SOURCES.txt'
reading manifest template 'MANIFEST.in'
warning: no previously-included files found matching 'third-party/zlib/Makefile'
warning: no previously-included files found matching 'third-party/zlib/zconf.h'
warning: no previously-included files matching '*.orig' found anywhere in distribution
warning: no previously-included files matching '*.pyc' found anywhere in distribution
writing manifest file 'khmer.egg-info/SOURCES.txt'
installing library code to build/bdist.linux-x86_64/egg
running install_lib
running build_py
creating build
creating build/lib.linux-x86_64-2.7
creating build/lib.linux-x86_64-2.7/khmer
copying khmer/kfile.py -> build/lib.linux-x86_64-2.7/khmer
copying khmer/khmer_args.py -> build/lib.linux-x86_64-2.7/khmer
copying khmer/thread_utils.py -> build/lib.linux-x86_64-2.7/khmer
copying khmer/utils.py -> build/lib.linux-x86_64-2.7/khmer
copying khmer/__init__.py -> build/lib.linux-x86_64-2.7/khmer
copying khmer/_version.py -> build/lib.linux-x86_64-2.7/khmer
creating build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_scripts.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_threaded_sequence_processor.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_functions.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_hll.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_counting_single.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_read_aligner.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_labelhash.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_normalize_by_median.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_subset_graph.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/__init__.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_counting_hash.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_hashbits.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_hashbits_obj.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_version.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_graph.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_filter.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/khmer_tst_utils.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_script_arguments.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_sandbox_scripts.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_read_parsers.py -> build/lib.linux-x86_64-2.7/khmer/tests
copying tests/test_lump.py -> build/lib.linux-x86_64-2.7/khmer/tests
creating build/lib.linux-x86_64-2.7/oxli
copying oxli/functions.py -> build/lib.linux-x86_64-2.7/oxli
copying oxli/build_graph.py -> build/lib.linux-x86_64-2.7/oxli
copying oxli/__init__.py -> build/lib.linux-x86_64-2.7/oxli
copying khmer/_khmer.cc -> build/lib.linux-x86_64-2.7/khmer
copying tests/.gitignore -> build/lib.linux-x86_64-2.7/khmer/tests
creating build/lib.linux-x86_64-2.7/khmer/tests/test-data
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copying tests/test-data/100-reads.fq.truncated.gz -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/all-A.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/badversion-k12.ct -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/badversion-k12.ht -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/badversion-k32.stoptags -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/badversion-k32.tagset -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/biglump-random-20-a.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/casava_18-pe.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/combine_parts_1.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/dn-test-all-paired-all-keep.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/dn-test-none-paired.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/dn-test-some-paired-all-keep.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/empty-file -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/fakelump.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/fakelump.fa.stoptags.txt -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
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copying tests/test-data/goodversion-k32.stoptags -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/goodversion-k32.tagset -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/normC20k20.ct -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/old-style-format-w-comments.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/overlap.out -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-broken.fq.1 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-broken.fq.2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-broken2.fq.1 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-broken2.fq.2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-broken3.fq.1 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-broken3.fq.2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-mixed-2.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-mixed-broken.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-mixed.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-mixed.fa.pe -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-mixed.fa.se -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-mixed.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-mixed.fq.pe -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired-mixed.fq.se -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired.fa.1 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired.fa.2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired.fq.1 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired.fq.2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/paired_one.base.dif.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-X2.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.even.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.fa.bz2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.fa.gz -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.fa.part -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.fq.bz2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.fq.gz -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-a.odd.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-20-b.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/random-31-c.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/real-partition-small.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/real-partition-tiny.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/single-read.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-2.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-2.fa.bz2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-2.fa.gz -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-2.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-2.paired.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-2.paired2.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-3.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-impaired.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-paired.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read-paired.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-abund-read.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-empty.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-empty.fa.bz2 -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-error-reads.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-fastq-n-reads.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-fastq-reads.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-filter-abund-Ns.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-graph.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-graph2.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-graph5.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-labels.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-large.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-output-partitions.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-overlap1.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-overlap2.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
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copying tests/test-data/test-reads.fq.gz -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-short.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-sweep-contigs.fp -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-sweep-reads.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-sweep-reads.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/test-transcript.fa -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
copying tests/test-data/truncated.fq -> build/lib.linux-x86_64-2.7/khmer/tests/test-data
running build_ext
bash -c cd third-party/zlib && ( test Makefile -nt configure || bash ./configure --static ) && make -f Makefile.pic PIC
Checking for gcc...
Building static library libz.a version 1.2.8 with gcc.
Checking for off64_t... Yes.
Checking for fseeko... Yes.
Checking for strerror... Yes.
Checking for unistd.h... Yes.
Checking for stdarg.h... Yes.
Checking whether to use vs[n]printf() or s[n]printf()... using vs[n]printf().
Checking for vsnprintf() in stdio.h... Yes.
Checking for return value of vsnprintf()... Yes.
Checking for attribute(visibility) support... Yes.
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/adler32.o adler32.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/crc32.o crc32.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/deflate.o deflate.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/infback.o infback.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/inffast.o inffast.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/inflate.o inflate.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/inftrees.o inftrees.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/trees.o trees.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/zutil.o zutil.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/compress.o compress.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/uncompr.o uncompr.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/gzclose.o gzclose.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/gzlib.o gzlib.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/gzread.o gzread.c
gcc -O3  -fPIC -D_LARGEFILE64_SOURCE=1 -DHAVE_HIDDEN -DPIC -c -o objs/gzwrite.o gzwrite.c
bash -c cd third-party/bzip2 && make -f Makefile-libbz2_so all
gcc -fpic -fPIC -Wall -Winline -O2 -g -D_FILE_OFFSET_BITS=64 -c blocksort.c
In function ‘mainSort’:
blocksort.c:347:6: warning: inlining failed in call to ‘mainGtU.part.0’: call is unlikely and code size would grow [-Winline]
 Bool mainGtU ( UInt32  i1, 
      ^
cc1: warning: called from here [-Winline]
blocksort.c:347:6: warning: inlining failed in call to ‘mainGtU.part.0’: call is unlikely and code size would grow [-Winline]
 Bool mainGtU ( UInt32  i1, 
      ^
cc1: warning: called from here [-Winline]
blocksort.c:347:6: warning: inlining failed in call to ‘mainGtU.part.0’: call is unlikely and code size would grow [-Winline]
 Bool mainGtU ( UInt32  i1, 
      ^
cc1: warning: called from here [-Winline]
gcc -fpic -fPIC -Wall -Winline -O2 -g -D_FILE_OFFSET_BITS=64 -c huffman.c
gcc -fpic -fPIC -Wall -Winline -O2 -g -D_FILE_OFFSET_BITS=64 -c crctable.c
gcc -fpic -fPIC -Wall -Winline -O2 -g -D_FILE_OFFSET_BITS=64 -c randtable.c
gcc -fpic -fPIC -Wall -Winline -O2 -g -D_FILE_OFFSET_BITS=64 -c compress.c
gcc -fpic -fPIC -Wall -Winline -O2 -g -D_FILE_OFFSET_BITS=64 -c decompress.c
gcc -fpic -fPIC -Wall -Winline -O2 -g -D_FILE_OFFSET_BITS=64 -c bzlib.c
#gcc -shared -Wl,-soname -Wl,libbz2.so.1.0 -o libbz2.so.1.0.6 blocksort.o huffman.o crctable.o randtable.o compress.o decompress.o bzlib.o
#gcc -fpic -fPIC -Wall -Winline -O2 -g -D_FILE_OFFSET_BITS=64 -o bzip2-shared bzip2.c libbz2.so.1.0.6
#rm -f libbz2.so.1.0
#ln -s libbz2.so.1.0.6 libbz2.so.1.0
building 'khmer._khmer' extension
creating build/temp.linux-x86_64-2.7
creating build/temp.linux-x86_64-2.7/khmer
creating build/temp.linux-x86_64-2.7/lib
creating build/temp.linux-x86_64-2.7/third-party
creating build/temp.linux-x86_64-2.7/third-party/smhasher
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c khmer/_khmer.cc -o build/temp.linux-x86_64-2.7/khmer/_khmer.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/trace_logger.cc -o build/temp.linux-x86_64-2.7/lib/trace_logger.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/perf_metrics.cc -o build/temp.linux-x86_64-2.7/lib/perf_metrics.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/read_parsers.cc -o build/temp.linux-x86_64-2.7/lib/read_parsers.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/kmer_hash.cc -o build/temp.linux-x86_64-2.7/lib/kmer_hash.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/hashtable.cc -o build/temp.linux-x86_64-2.7/lib/hashtable.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/hashbits.cc -o build/temp.linux-x86_64-2.7/lib/hashbits.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/labelhash.cc -o build/temp.linux-x86_64-2.7/lib/labelhash.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/counting.cc -o build/temp.linux-x86_64-2.7/lib/counting.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/subset.cc -o build/temp.linux-x86_64-2.7/lib/subset.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/read_aligner.cc -o build/temp.linux-x86_64-2.7/lib/read_aligner.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c lib/hllcounter.cc -o build/temp.linux-x86_64-2.7/lib/hllcounter.o -O3 -fopenmp
x86_64-linux-gnu-gcc -pthread -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -fPIC -DVERSION=1.4+261.g75f6fbf -DSEQAN_HAS_BZIP2=1 -DSEQAN_HAS_ZLIB=1 -UNO_UNIQUE_RC -Ilib -Ithird-party/zlib -Ithird-party/bzip2 -Ithird-party/seqan/core/include -Ithird-party/smhasher -I/usr/include/python2.7 -c third-party/smhasher/MurmurHash3.cc -o build/temp.linux-x86_64-2.7/third-party/smhasher/MurmurHash3.o -O3 -fopenmp
c++ -pthread -shared -Wl,-O1 -Wl,-Bsymbolic-functions -Wl,-Bsymbolic-functions -Wl,-z,relro -fno-strict-aliasing -DNDEBUG -g -fwrapv -O2 -Wall -Wstrict-prototypes -D_FORTIFY_SOURCE=2 -g -fstack-protector --param=ssp-buffer-size=4 -Wformat -Werror=format-security build/temp.linux-x86_64-2.7/khmer/_khmer.o build/temp.linux-x86_64-2.7/lib/trace_logger.o build/temp.linux-x86_64-2.7/lib/perf_metrics.o build/temp.linux-x86_64-2.7/lib/read_parsers.o build/temp.linux-x86_64-2.7/lib/kmer_hash.o build/temp.linux-x86_64-2.7/lib/hashtable.o build/temp.linux-x86_64-2.7/lib/hashbits.o build/temp.linux-x86_64-2.7/lib/labelhash.o build/temp.linux-x86_64-2.7/lib/counting.o build/temp.linux-x86_64-2.7/lib/subset.o build/temp.linux-x86_64-2.7/lib/read_aligner.o build/temp.linux-x86_64-2.7/lib/hllcounter.o build/temp.linux-x86_64-2.7/third-party/smhasher/MurmurHash3.o third-party/zlib/adler32.lo third-party/zlib/compress.lo third-party/zlib/crc32.lo third-party/zlib/deflate.lo third-party/zlib/gzclose.lo third-party/zlib/gzlib.lo third-party/zlib/gzread.lo third-party/zlib/gzwrite.lo third-party/zlib/infback.lo third-party/zlib/inffast.lo third-party/zlib/inflate.lo third-party/zlib/inftrees.lo third-party/zlib/trees.lo third-party/zlib/uncompr.lo third-party/zlib/zutil.lo third-party/bzip2/blocksort.o third-party/bzip2/huffman.o third-party/bzip2/crctable.o third-party/bzip2/randtable.o third-party/bzip2/compress.o third-party/bzip2/decompress.o third-party/bzip2/bzlib.o -o build/lib.linux-x86_64-2.7/khmer/_khmer.so -fopenmp
creating build/bdist.linux-x86_64
creating build/bdist.linux-x86_64/egg
creating build/bdist.linux-x86_64/egg/khmer
copying build/lib.linux-x86_64-2.7/khmer/_khmer.cc -> build/bdist.linux-x86_64/egg/khmer
copying build/lib.linux-x86_64-2.7/khmer/kfile.py -> build/bdist.linux-x86_64/egg/khmer
copying build/lib.linux-x86_64-2.7/khmer/khmer_args.py -> build/bdist.linux-x86_64/egg/khmer
copying build/lib.linux-x86_64-2.7/khmer/thread_utils.py -> build/bdist.linux-x86_64/egg/khmer
copying build/lib.linux-x86_64-2.7/khmer/utils.py -> build/bdist.linux-x86_64/egg/khmer
copying build/lib.linux-x86_64-2.7/khmer/__init__.py -> build/bdist.linux-x86_64/egg/khmer
creating build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_scripts.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_threaded_sequence_processor.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/.gitignore -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_functions.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_hll.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_counting_single.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_read_aligner.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_labelhash.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_normalize_by_median.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_subset_graph.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/__init__.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_counting_hash.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_hashbits.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_hashbits_obj.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_version.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_graph.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_filter.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/khmer_tst_utils.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_script_arguments.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_sandbox_scripts.py -> build/bdist.linux-x86_64/egg/khmer/tests
creating build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/badversion-k32.tagset -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/goodversion-k32.tagset -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/100-reads.fq.truncated.bz2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/badversion-k32.stoptags -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/dn-test-all-paired-all-keep.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/biglump-random-20-a.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-3.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/badversion-k12.ht -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired.fq.2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.fq.gz -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-mixed.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/fakelump.fa.stoptags.txt -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-mixed.fa.se -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/overlap.out -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/goodversion-k12.ht.gz -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-broken.fq.2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-broken3.fq.2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.odd.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-sweep-reads.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired.fq.1 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-graph5.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-fastq-n-reads.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-paired.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-sweep-contigs.fp -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-output-partitions.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.fa.bz2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-mixed.fq.se -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/dn-test-some-paired-all-keep.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/combine_parts_1.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-mixed-broken.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/filter-test-A.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-sweep-reads.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-X2.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-paired.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-overlap2.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/normC20k20.ct -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/goodversion-k12.ht -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-reads.fq.gz -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/100-reads.fq.gz -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired.fa.1 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/old-style-format-w-comments.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-2.paired.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-empty.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-fastq-reads.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-2.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-filter-abund-Ns.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/dn-test-none-paired.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.fa.part -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-broken2.fq.2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/single-read.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-labels.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-empty.fa.bz2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-reads.fq.bz2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-mixed.fq.pe -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/empty-file -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/100-reads.fq.truncated.gz -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/real-partition-tiny.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-large.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/real-partition-small.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-transcript.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-mixed.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-overlap1.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired.fa.2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-impaired.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.fa.gz -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/casava_18-pe.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-2.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-graph2.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-broken3.fq.1 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-graph.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.fq.bz2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/fakelump.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired_one.base.dif.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-mixed-2.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-2.paired2.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-short.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/100-reads.fq.bz2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-reads.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-2.fa.gz -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-b.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-error-reads.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/badversion-k12.ct -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/test-abund-read-2.fa.bz2 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/all-A.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-broken.fq.1 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/filter-test-B.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/goodversion-k32.stoptags -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-20-a.even.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/truncated.fq -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-mixed.fa.pe -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/paired-broken2.fq.1 -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test-data/random-31-c.fa -> build/bdist.linux-x86_64/egg/khmer/tests/test-data
copying build/lib.linux-x86_64-2.7/khmer/tests/test_read_parsers.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/tests/test_lump.py -> build/bdist.linux-x86_64/egg/khmer/tests
copying build/lib.linux-x86_64-2.7/khmer/_khmer.so -> build/bdist.linux-x86_64/egg/khmer
copying build/lib.linux-x86_64-2.7/khmer/_version.py -> build/bdist.linux-x86_64/egg/khmer
creating build/bdist.linux-x86_64/egg/oxli
copying build/lib.linux-x86_64-2.7/oxli/functions.py -> build/bdist.linux-x86_64/egg/oxli
copying build/lib.linux-x86_64-2.7/oxli/build_graph.py -> build/bdist.linux-x86_64/egg/oxli
copying build/lib.linux-x86_64-2.7/oxli/__init__.py -> build/bdist.linux-x86_64/egg/oxli
byte-compiling build/bdist.linux-x86_64/egg/khmer/kfile.py to kfile.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/khmer_args.py to khmer_args.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/thread_utils.py to thread_utils.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/utils.py to utils.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/__init__.py to __init__.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_scripts.py to test_scripts.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_threaded_sequence_processor.py to test_threaded_sequence_processor.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_functions.py to test_functions.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_hll.py to test_hll.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_counting_single.py to test_counting_single.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_read_aligner.py to test_read_aligner.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_labelhash.py to test_labelhash.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_normalize_by_median.py to test_normalize_by_median.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_subset_graph.py to test_subset_graph.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/__init__.py to __init__.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_counting_hash.py to test_counting_hash.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_hashbits.py to test_hashbits.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_hashbits_obj.py to test_hashbits_obj.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_version.py to test_version.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_graph.py to test_graph.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_filter.py to test_filter.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/khmer_tst_utils.py to khmer_tst_utils.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_script_arguments.py to test_script_arguments.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_sandbox_scripts.py to test_sandbox_scripts.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_read_parsers.py to test_read_parsers.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/tests/test_lump.py to test_lump.pyc
byte-compiling build/bdist.linux-x86_64/egg/khmer/_version.py to _version.pyc
byte-compiling build/bdist.linux-x86_64/egg/oxli/functions.py to functions.pyc
byte-compiling build/bdist.linux-x86_64/egg/oxli/build_graph.py to build_graph.pyc
byte-compiling build/bdist.linux-x86_64/egg/oxli/__init__.py to __init__.pyc
creating stub loader for khmer/_khmer.so
byte-compiling build/bdist.linux-x86_64/egg/khmer/_khmer.py to _khmer.pyc
creating build/bdist.linux-x86_64/egg/EGG-INFO
installing scripts to build/bdist.linux-x86_64/egg/EGG-INFO/scripts
running install_scripts
running build_scripts
creating build/scripts-2.7
copying and adjusting scripts/normalize-by-median.py -> build/scripts-2.7
copying and adjusting scripts/load-graph.py -> build/scripts-2.7
copying and adjusting scripts/abundance-dist.py -> build/scripts-2.7
copying and adjusting scripts/annotate-partitions.py -> build/scripts-2.7
copying and adjusting scripts/abundance-dist-single.py -> build/scripts-2.7
copying and adjusting scripts/extract-long-sequences.py -> build/scripts-2.7
copying and adjusting scripts/count-overlap.py -> build/scripts-2.7
copying and adjusting scripts/partition-graph.py -> build/scripts-2.7
copying and adjusting scripts/merge-partitions.py -> build/scripts-2.7
copying and adjusting scripts/trim-low-abund.py -> build/scripts-2.7
copying and adjusting scripts/readstats.py -> build/scripts-2.7
copying and adjusting scripts/do-partition.py -> build/scripts-2.7
copying and adjusting scripts/load-into-counting.py -> build/scripts-2.7
copying and adjusting scripts/fastq-to-fasta.py -> build/scripts-2.7
copying and adjusting scripts/sample-reads-randomly.py -> build/scripts-2.7
copying and adjusting scripts/find-knots.py -> build/scripts-2.7
copying and adjusting scripts/filter-stoptags.py -> build/scripts-2.7
copying and adjusting scripts/count-median.py -> build/scripts-2.7
copying and adjusting scripts/extract-paired-reads.py -> build/scripts-2.7
copying and adjusting scripts/interleave-reads.py -> build/scripts-2.7
copying and adjusting scripts/extract-partitions.py -> build/scripts-2.7
copying and adjusting scripts/split-paired-reads.py -> build/scripts-2.7
copying and adjusting scripts/filter-abund.py -> build/scripts-2.7
copying and adjusting scripts/make-initial-stoptags.py -> build/scripts-2.7
copying and adjusting scripts/filter-abund-single.py -> build/scripts-2.7
changing mode of build/scripts-2.7/normalize-by-median.py from 644 to 755
changing mode of build/scripts-2.7/load-graph.py from 644 to 755
changing mode of build/scripts-2.7/abundance-dist.py from 644 to 755
changing mode of build/scripts-2.7/annotate-partitions.py from 644 to 755
changing mode of build/scripts-2.7/abundance-dist-single.py from 644 to 755
changing mode of build/scripts-2.7/extract-long-sequences.py from 644 to 755
changing mode of build/scripts-2.7/count-overlap.py from 644 to 755
changing mode of build/scripts-2.7/partition-graph.py from 644 to 755
changing mode of build/scripts-2.7/merge-partitions.py from 644 to 755
changing mode of build/scripts-2.7/trim-low-abund.py from 644 to 755
changing mode of build/scripts-2.7/readstats.py from 644 to 755
changing mode of build/scripts-2.7/do-partition.py from 644 to 755
changing mode of build/scripts-2.7/load-into-counting.py from 644 to 755
changing mode of build/scripts-2.7/fastq-to-fasta.py from 644 to 755
changing mode of build/scripts-2.7/sample-reads-randomly.py from 644 to 755
changing mode of build/scripts-2.7/find-knots.py from 644 to 755
changing mode of build/scripts-2.7/filter-stoptags.py from 644 to 755
changing mode of build/scripts-2.7/count-median.py from 644 to 755
changing mode of build/scripts-2.7/extract-paired-reads.py from 644 to 755
changing mode of build/scripts-2.7/interleave-reads.py from 644 to 755
changing mode of build/scripts-2.7/extract-partitions.py from 644 to 755
changing mode of build/scripts-2.7/split-paired-reads.py from 644 to 755
changing mode of build/scripts-2.7/filter-abund.py from 644 to 755
changing mode of build/scripts-2.7/make-initial-stoptags.py from 644 to 755
changing mode of build/scripts-2.7/filter-abund-single.py from 644 to 755
creating build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/normalize-by-median.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/load-graph.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/abundance-dist.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/annotate-partitions.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/abundance-dist-single.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/extract-long-sequences.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/count-overlap.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/partition-graph.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/merge-partitions.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/trim-low-abund.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/readstats.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/do-partition.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/load-into-counting.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/fastq-to-fasta.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/sample-reads-randomly.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/find-knots.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/filter-stoptags.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/count-median.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/extract-paired-reads.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/interleave-reads.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/extract-partitions.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/split-paired-reads.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/filter-abund.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/make-initial-stoptags.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/filter-abund-single.py -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
changing mode of build/bdist.linux-x86_64/egg/EGG-INFO/scripts/normalize-by-median.py to 755
changing mode of build/bdist.linux-x86_64/egg/EGG-INFO/scripts/load-graph.py to 755
changing mode of build/bdist.linux-x86_64/egg/EGG-INFO/scripts/abundance-dist.py to 755
changing mode of build/bdist.linux-x86_64/egg/EGG-INFO/scripts/annotate-partitions.py to 755
changing mode of build/bdist.linux-x86_64/egg/EGG-INFO/scripts/abundance-dist-single.py to 755
changing mode of build/bdist.linux-x86_64/egg/EGG-INFO/scripts/extract-long-sequences.py to 755
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The following script is contained within the khmer package and can estimate the total unique number of k-mers in your dataset. Use cases for this might be a) determining how diverse a metagenome is compared to e.g., a bacterial genome for assembly, b) to compare k-mer diversity among multiple metagenomes, c) exploring the impacts of choice of length k for assembly.

Next, to estimate the number unique k-mers in the datasets for multiple k's (17, 21, 25, 29, 33, 37), execute the scripts below. The script output will identify the unique k-mers but will also save in a report named unique_count. (This takes about 15 minutes on a large instance and 8-10 minutes on an extra large.)


In [20]:
!python unique-kmers.py -R unique_count -k 17 SRR172903.qc.fastq
!python unique-kmers.py -R unique_count -k 21 SRR172903.qc.fastq
!python unique-kmers.py -R unique_count -k 25 SRR172903.qc.fastq
!python unique-kmers.py -R unique_count -k 29 SRR172903.qc.fastq
!python unique-kmers.py -R unique_count -k 33 SRR172903.qc.fastq
!python unique-kmers.py -R unique_count -k 37 SRR172903.qc.fastq


|| This is the script 'unique-kmers.py' in khmer.
|| You are running khmer version 0+unknown
|| You are also using screed version 0.9
||
|| If you use this script in a publication, please cite EACH of the following:
||
||   * MR Crusoe et al., 2014. http://dx.doi.org/10.6084/m9.figshare.979190
||   * A. Döring et al. http://dx.doi.org:80/10.1186/1471-2105-9-11
||   * Irber and Brown, unpublished
||
|| Please see http://khmer.readthedocs.org/en/latest/citations.html for details.

Estimated number of unique k-mers: 14330058

|| This is the script 'unique-kmers.py' in khmer.
|| You are running khmer version 0+unknown
|| You are also using screed version 0.9
||
|| If you use this script in a publication, please cite EACH of the following:
||
||   * MR Crusoe et al., 2014. http://dx.doi.org/10.6084/m9.figshare.979190
||   * A. Döring et al. http://dx.doi.org:80/10.1186/1471-2105-9-11
||   * Irber and Brown, unpublished
||
|| Please see http://khmer.readthedocs.org/en/latest/citations.html for details.

Estimated number of unique k-mers: 15287036

|| This is the script 'unique-kmers.py' in khmer.
|| You are running khmer version 0+unknown
|| You are also using screed version 0.9
||
|| If you use this script in a publication, please cite EACH of the following:
||
||   * MR Crusoe et al., 2014. http://dx.doi.org/10.6084/m9.figshare.979190
||   * A. Döring et al. http://dx.doi.org:80/10.1186/1471-2105-9-11
||   * Irber and Brown, unpublished
||
|| Please see http://khmer.readthedocs.org/en/latest/citations.html for details.

Estimated number of unique k-mers: 15446874

|| This is the script 'unique-kmers.py' in khmer.
|| You are running khmer version 0+unknown
|| You are also using screed version 0.9
||
|| If you use this script in a publication, please cite EACH of the following:
||
||   * MR Crusoe et al., 2014. http://dx.doi.org/10.6084/m9.figshare.979190
||   * A. Döring et al. http://dx.doi.org:80/10.1186/1471-2105-9-11
||   * Irber and Brown, unpublished
||
|| Please see http://khmer.readthedocs.org/en/latest/citations.html for details.

Estimated number of unique k-mers: 15427477

|| This is the script 'unique-kmers.py' in khmer.
|| You are running khmer version 0+unknown
|| You are also using screed version 0.9
||
|| If you use this script in a publication, please cite EACH of the following:
||
||   * MR Crusoe et al., 2014. http://dx.doi.org/10.6084/m9.figshare.979190
||   * A. Döring et al. http://dx.doi.org:80/10.1186/1471-2105-9-11
||   * Irber and Brown, unpublished
||
|| Please see http://khmer.readthedocs.org/en/latest/citations.html for details.

Estimated number of unique k-mers: 15147325

|| This is the script 'unique-kmers.py' in khmer.
|| You are running khmer version 0+unknown
|| You are also using screed version 0.9
||
|| If you use this script in a publication, please cite EACH of the following:
||
||   * MR Crusoe et al., 2014. http://dx.doi.org/10.6084/m9.figshare.979190
||   * A. Döring et al. http://dx.doi.org:80/10.1186/1471-2105-9-11
||   * Irber and Brown, unpublished
||
|| Please see http://khmer.readthedocs.org/en/latest/citations.html for details.

Estimated number of unique k-mers: 14910222

You can see that this file now has in the first column the k-mer length and in the second column the estimated number of words of length k in the metagenomes. If you had multiple genomes, you could compare diversity of e.g., the total number of k-mers across datasets. To view the results of the file, you can use the concatentate program/command "cat".


In [21]:
!cat unique_count


17 14330058
21 15287036
25 15446874
29 15427477
33 15147325
37 14910222

5. Getting a sequence coverage profile: What genes are present in my metagenome?

Most metagenomic analysis require one to estimate the abundance of reference genes (e.g., orginating from genomes or one's own metagenomic assembly). This tutorial will cover both cases where references are available or unavailable (requiring de novo assembly).

6. Case I - Reference genomes available.

For the mock HMP metagenome, the HMP has sequenced the genomes of the isolates used for this simulated dataset. The list of these genomes can be obtained on the HMP website, and we provide it here in a Github repository, a tool used for collaboratively sharing data and code. The command below will download data for this tutorial.


In [22]:
!cat ncbi_acc.txt


NC_005008.1
NC_005007.1
NC_005003.1
NC_005006.1
NC_005004.1
NC_009084.1
NC_005005.1
NC_000958.1
NC_000959.1
NC_009083.1
NC_001264.1
NC_001263.1
NC_004461.1
NC_009008.1
NC_010079.1
NC_007490.1
NC_009007.1
NC_007489.1
NC_004350.1
NC_007488.1
NC_007493.1
NC_007494.1
NC_009085.1
NC_009515.1
NC_009614.1
NC_000915.1
NC_003028.3
NC_000913.2
NC_006085.1
NC_003112.2

The following command downloads all the genomes for each ID in the above list into a directory called "genomes".


In [23]:
!python fetch-genomes-fasta.py ncbi_acc.txt genomes


Fetching NC_005008.1...Done
Fetching NC_005007.1...Done
Fetching NC_005003.1...Done
Fetching NC_005006.1...Done
Fetching NC_005004.1...Done
Fetching NC_009084.1...Done
Fetching NC_005005.1...Done
Fetching NC_000958.1...Done
Fetching NC_000959.1...Done
Fetching NC_009083.1...Done
Fetching NC_001264.1...Done
Fetching NC_001263.1...Done
Fetching NC_004461.1...Done
Fetching NC_009008.1...Done
Fetching NC_010079.1...Done
Fetching NC_007490.1...Done
Fetching NC_009007.1...Done
Fetching NC_007489.1...Done
Fetching NC_004350.1...Done
Fetching NC_007488.1...Done
Fetching NC_007493.1...Done
Fetching NC_007494.1...Done
Fetching NC_009085.1...Done
Fetching NC_009515.1...Done
Fetching NC_009614.1...Done
Fetching NC_000915.1...Done
Fetching NC_003028.3...Done
Fetching NC_000913.2...Done
Fetching NC_006085.1...Done
Fetching NC_003112.2...Done

7. Estimating abundance of assembled contigs

To estimate the representation of reference genes or genomes in your metagenome, you can align reads to references using read mapping software (e.g., Bowtie2, BWA, etc.). In this tutorial, we will use Bowtie2 which we will install on this server. We will then be mapping the metagenome to a single reference genome (that we downloaded above).


In [24]:
!wget http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.5/bowtie2-2.2.5-linux-x86_64.zip
!unzip bowtie2-2.2.5-linux-x86_64.zip


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Location: http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.5/bowtie2-2.2.5-linux-x86_64.zip/download [following]
--2015-06-26 14:07:44--  http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.5/bowtie2-2.2.5-linux-x86_64.zip/download
Connecting to sourceforge.net (sourceforge.net)|216.34.181.60|:80... connected.
HTTP request sent, awaiting response... 302 Found
Location: http://downloads.sourceforge.net/project/bowtie-bio/bowtie2/2.2.5/bowtie2-2.2.5-linux-x86_64.zip?r=&ts=1435327664&use_mirror=iweb [following]
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Resolving downloads.sourceforge.net (downloads.sourceforge.net)... 216.34.181.59
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HTTP request sent, awaiting response... 302 Found
Location: http://iweb.dl.sourceforge.net/project/bowtie-bio/bowtie2/2.2.5/bowtie2-2.2.5-linux-x86_64.zip [following]
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Resolving iweb.dl.sourceforge.net (iweb.dl.sourceforge.net)... 70.38.0.134, 2607:f748:10:12::5f:2
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HTTP request sent, awaiting response... 200 OK
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Saving to: ‘bowtie2-2.2.5-linux-x86_64.zip’

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I've written a script that will automatically map a set of reads to a given reference and output a file containing the number of reads that are mapped to a given reference. To use this script, we'll also need to install samtools. A samfile is a super compressed file that efficiently stores mapped information from mappers. Samtools helps us interact with this file.


In [25]:
!apt-get install samtools




The following NEW packages will be installed:
  samtools
0 upgraded, 1 newly installed, 0 to remove and 14 not upgraded.
Need to get 560 kB of archives.
After this operation, 1,336 kB of additional disk space will be used.
Get:1 http://us-east-1.ec2.archive.ubuntu.com/ubuntu/ saucy/universe samtools amd64 0.1.19-1 [560 kB]
Fetched 560 kB in 0s (22.3 MB/s)
Selecting previously unselected package samtools.
(Reading database ... 112754 files and directories currently installed.)
Unpacking samtools (from .../samtools_0.1.19-1_amd64.deb) ...
Processing triggers for man-db ...
Setting up samtools (0.1.19-1) ...

To map reads to a reference, we have provided an easy to use program. The steps the program performs are as follows:

  • Index your reference genome,
  • Map reads to your index genome (with default bowtie parameters),
  • Use Samtools to estimate the number of reads mapped, number of reads unmapped, and provide a tab delimited file with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads.

This takes about 8-10 minutes.


In [26]:
!bash bowtie.sh genomes/NC_000913.2.fa SRR172903.qc.fastq


Reference is genomes/NC_000913.2.fa
Reads are SRR172903.qc.fastq
Settings:
  Output files: "genomes/NC_000913.2.fa-bowtie-index.*.bt2"
  Line rate: 6 (line is 64 bytes)
  Lines per side: 1 (side is 64 bytes)
  Offset rate: 4 (one in 16)
  FTable chars: 10
  Strings: unpacked
  Max bucket size: default
  Max bucket size, sqrt multiplier: default
  Max bucket size, len divisor: 4
  Difference-cover sample period: 1024
  Endianness: little
  Actual local endianness: little
  Sanity checking: disabled
  Assertions: disabled
  Random seed: 0
  Sizeofs: void*:8, int:4, long:8, size_t:8
Input files DNA, FASTA:
  genomes/NC_000913.2.fa
Building a SMALL index
Reading reference sizes
  Time reading reference sizes: 00:00:00
Calculating joined length
Writing header
Reserving space for joined string
Joining reference sequences
  Time to join reference sequences: 00:00:00
bmax according to bmaxDivN setting: 1159918
Using parameters --bmax 869939 --dcv 1024
  Doing ahead-of-time memory usage test
  Passed!  Constructing with these parameters: --bmax 869939 --dcv 1024
Constructing suffix-array element generator
Building DifferenceCoverSample
  Building sPrime
  Building sPrimeOrder
  V-Sorting samples
  V-Sorting samples time: 00:00:00
  Allocating rank array
  Ranking v-sort output
  Ranking v-sort output time: 00:00:00
  Invoking Larsson-Sadakane on ranks
  Invoking Larsson-Sadakane on ranks time: 00:00:00
  Sanity-checking and returning
Building samples
Reserving space for 12 sample suffixes
Generating random suffixes
QSorting 12 sample offsets, eliminating duplicates
QSorting sample offsets, eliminating duplicates time: 00:00:00
Multikey QSorting 12 samples
  (Using difference cover)
  Multikey QSorting samples time: 00:00:00
Calculating bucket sizes
  Binary sorting into buckets
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  Binary sorting into buckets time: 00:00:00
Splitting and merging
  Splitting and merging time: 00:00:00
Split 2, merged 6; iterating...
  Binary sorting into buckets
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  Binary sorting into buckets time: 00:00:00
Splitting and merging
  Splitting and merging time: 00:00:00
Split 1, merged 1; iterating...
  Binary sorting into buckets
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  Binary sorting into buckets time: 00:00:01
Splitting and merging
  Splitting and merging time: 00:00:00
Avg bucket size: 579958 (target: 869938)
Converting suffix-array elements to index image
Allocating ftab, absorbFtab
Entering Ebwt loop
Getting block 1 of 8
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 566788
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 566789
Getting block 2 of 8
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 564637
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 564638
Getting block 3 of 8
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 645098
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 645099
Getting block 4 of 8
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 234151
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 234152
Getting block 5 of 8
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 643592
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 643593
Getting block 6 of 8
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 711613
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 711614
Getting block 7 of 8
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 574603
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 574604
Getting block 8 of 8
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 699186
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 699187
Exited Ebwt loop
fchr[A]: 0
fchr[C]: 1142228
fchr[G]: 2321782
fchr[T]: 3498705
fchr[$]: 4639675
Exiting Ebwt::buildToDisk()
Returning from initFromVector
Wrote 5741113 bytes to primary EBWT file: genomes/NC_000913.2.fa-bowtie-index.1.bt2
Wrote 1159924 bytes to secondary EBWT file: genomes/NC_000913.2.fa-bowtie-index.2.bt2
Re-opening _in1 and _in2 as input streams
Returning from Ebwt constructor
Headers:
    len: 4639675
    bwtLen: 4639676
    sz: 1159919
    bwtSz: 1159919
    lineRate: 6
    offRate: 4
    offMask: 0xfffffff0
    ftabChars: 10
    eftabLen: 20
    eftabSz: 80
    ftabLen: 1048577
    ftabSz: 4194308
    offsLen: 289980
    offsSz: 1159920
    lineSz: 64
    sideSz: 64
    sideBwtSz: 48
    sideBwtLen: 192
    numSides: 24165
    numLines: 24165
    ebwtTotLen: 1546560
    ebwtTotSz: 1546560
    color: 0
    reverse: 0
Total time for call to driver() for forward index: 00:00:02
Reading reference sizes
  Time reading reference sizes: 00:00:00
Calculating joined length
Writing header
Reserving space for joined string
Joining reference sequences
  Time to join reference sequences: 00:00:00
  Time to reverse reference sequence: 00:00:00
bmax according to bmaxDivN setting: 1159918
Using parameters --bmax 869939 --dcv 1024
  Doing ahead-of-time memory usage test
  Passed!  Constructing with these parameters: --bmax 869939 --dcv 1024
Constructing suffix-array element generator
Building DifferenceCoverSample
  Building sPrime
  Building sPrimeOrder
  V-Sorting samples
  V-Sorting samples time: 00:00:00
  Allocating rank array
  Ranking v-sort output
  Ranking v-sort output time: 00:00:00
  Invoking Larsson-Sadakane on ranks
  Invoking Larsson-Sadakane on ranks time: 00:00:00
  Sanity-checking and returning
Building samples
Reserving space for 12 sample suffixes
Generating random suffixes
QSorting 12 sample offsets, eliminating duplicates
QSorting sample offsets, eliminating duplicates time: 00:00:00
Multikey QSorting 12 samples
  (Using difference cover)
  Multikey QSorting samples time: 00:00:00
Calculating bucket sizes
  Binary sorting into buckets
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  Binary sorting into buckets time: 00:00:01
Splitting and merging
  Splitting and merging time: 00:00:00
Split 2, merged 6; iterating...
  Binary sorting into buckets
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  Binary sorting into buckets time: 00:00:00
Splitting and merging
  Splitting and merging time: 00:00:00
Avg bucket size: 662810 (target: 869938)
Converting suffix-array elements to index image
Allocating ftab, absorbFtab
Entering Ebwt loop
Getting block 1 of 7
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 504667
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 504668
Getting block 2 of 7
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 574835
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 574836
Getting block 3 of 7
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 367744
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 367745
Getting block 4 of 7
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 868288
  (Using difference cover)
  Sorting block time: 00:00:01
Returning block of 868289
Getting block 5 of 7
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 852473
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 852474
Getting block 6 of 7
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 759657
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 759658
Getting block 7 of 7
  Reserving size (869939) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 712005
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 712006
Exited Ebwt loop
fchr[A]: 0
fchr[C]: 1142228
fchr[G]: 2321782
fchr[T]: 3498705
fchr[$]: 4639675
Exiting Ebwt::buildToDisk()
Returning from initFromVector
Wrote 5741113 bytes to primary EBWT file: genomes/NC_000913.2.fa-bowtie-index.rev.1.bt2
Wrote 1159924 bytes to secondary EBWT file: genomes/NC_000913.2.fa-bowtie-index.rev.2.bt2
Re-opening _in1 and _in2 as input streams
Returning from Ebwt constructor
Headers:
    len: 4639675
    bwtLen: 4639676
    sz: 1159919
    bwtSz: 1159919
    lineRate: 6
    offRate: 4
    offMask: 0xfffffff0
    ftabChars: 10
    eftabLen: 20
    eftabSz: 80
    ftabLen: 1048577
    ftabSz: 4194308
    offsLen: 289980
    offsSz: 1159920
    lineSz: 64
    sideSz: 64
    sideBwtSz: 48
    sideBwtLen: 192
    numSides: 24165
    numLines: 24165
    ebwtTotLen: 1546560
    ebwtTotSz: 1546560
    color: 0
    reverse: 1
Total time for backward call to driver() for mirror index: 00:00:02
820417 reads; of these:
  820417 (100.00%) were unpaired; of these:
    793072 (96.67%) aligned 0 times
    26531 (3.23%) aligned exactly 1 time
    814 (0.10%) aligned >1 times
3.33% overall alignment rate
[samopen] SAM header is present: 1 sequences.

We can look at the total number of reads mapped and unmapped from our metagenome to the genome NC_000913.2. We can also get a file that shows the reference sequence name (first column), reference sequence length (second column), # mapped reads (third column) and # unmapped reads (last column). The other columns contain information that samtools can use for other queries, you can read about samtools here, http://samtools.sourceforge.net/samtools.shtml.


In [27]:
!cat reads-mapped.count.txt


27345

In [28]:
!cat reads-unmapped.count.txt


793072

In [29]:
!cat reads.by.contigs.txt


gi|49175990|ref|NC_000913.2|	4639675	27345	0
*	0	0	793072

If you want a challenge, you can try mapping the metagenome to all reference genomes provided in the genome folder. To do so, try concatentating all genomes into one file (using this command: "cat genomes/*fa >> all-genomes.fa") and running the program on all-genomes.fa instead of NC_000913.2.fa.

8. Case II - De novo assembly of reference genes.

Assembly of the HMP mock metagenome

Assembly is the process of merging overlapping metagenomic reads from hopefully the same genome into a longer, continguous sequence (most commonly called a contig). It is advantageous in that it provides longer lengths for sequences that can later be used as references (that may be previously unknown), reduces the dataset size for analysis, and provides references that are not dependent on previous knowledge.

The choice of what assembler to use is not an easy one and is a subject of debate (see http://assemblathon.org). It is most important to remember that an assembly is a hypothesized consensus representation of your dataset. The assembly itself is an initial step that needs to be followed by an evaluation of its accuracy and usefulness. For most assemblers, the inputs are sequencing reads and paramters for the assembly software. For this tutorial, we will be completing the assembly with an assembler published in 2014 called Megahit (Li et al., 2015, https://github.com/voutcn/megahit). Sharpton's review (Sharpton, 2014) also reviews quite nicely some of the many assembly programs and approaches for metagenomic assembly.

To reduce the memory that is needed, it is often advantageous to normalize the distribution of k-mers in a metagenome. Removing extraneous information not needed for assembly also removes reads that may contain errors and may improve assembly (http://arxiv.org/abs/1203.4802). These scripts and tutorials are available at http://ged.msu.edu/angus/diginorm-2012/tutorial.html.

For this tutorial, we will assemble our metagenome with the Megathit assembler so first we have to install it.


In [30]:
!bash install-megahit.sh


Cloning into 'megahit'...
remote: Counting objects: 1505, done.
remote: Compressing objects: 100% (27/27), done.
remote: Total 1505 (delta 9), reused 0 (delta 0), pack-reused 1478
Receiving objects: 100% (1505/1505), 1.06 MiB | 0 bytes/s, done.
Resolving deltas: 100% (1033/1033), done.
Checking connectivity... done
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c succinct_dbg.cpp -o succinct_dbg.o
succinct_dbg.cpp: In member function ‘void SuccinctDBG::LoadFromFile(const char*)’:
succinct_dbg.cpp:369:34: warning: ignoring return value of ‘int fscanf(FILE*, const char*, ...)’, declared with attribute warn_unused_result [-Wunused-result]
     fscanf(f_file, "%d", &kmer_k);
                                  ^
succinct_dbg.cpp:370:45: warning: ignoring return value of ‘int fscanf(FILE*, const char*, ...)’, declared with attribute warn_unused_result [-Wunused-result]
     fscanf(f_file, "%u", &num_dollar_nodes_);
                                             ^
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c assembly_algorithms.cpp -o assembly_algorithms.o
In file included from hash_map.h:17:0,
                 from unitig_graph.h:30,
                 from assembly_algorithms.cpp:34:
hash_table.h: In instantiation of ‘struct HashTableNode<std::pair<long int, unsigned int> >’:
hash_table.h:599:39:   required from ‘void HashTable<Value, Key, HashFunc, ExtractKey, EqualKey>::clear() [with Value = std::pair<long int, unsigned int>; Key = long int; HashFunc = Hash<long int>; ExtractKey = Select1st<std::pair<long int, unsigned int> >; EqualKey = std::equal_to<long int>]’
hash_table.h:277:15:   required from ‘HashTable<Value, Key, HashFunc, ExtractKey, EqualKey>::~HashTable() [with Value = std::pair<long int, unsigned int>; Key = long int; HashFunc = Hash<long int>; ExtractKey = Select1st<std::pair<long int, unsigned int> >; EqualKey = std::equal_to<long int>]’
hash_map.h:32:7:   required from here
hash_table.h:50:7: warning: ignoring packed attribute because of unpacked non-POD field ‘std::pair<long int, unsigned int> HashTableNode<std::pair<long int, unsigned int> >::value’ [enabled by default]
     T value;
       ^
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c branch_group.cpp -o branch_group.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c options_description.cpp -o options_description.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c unitig_graph.cpp -o unitig_graph.o
In file included from hash_map.h:17:0,
                 from unitig_graph.h:30,
                 from unitig_graph.cpp:21:
hash_table.h: In instantiation of ‘struct HashTableNode<std::pair<long int, unsigned int> >’:
hash_table.h:102:16:   required from ‘HashTableIterator<Value, Key, HashFunc, ExtractKey, EqualKey>::value_type* HashTableIterator<Value, Key, HashFunc, ExtractKey, EqualKey>::operator->() const [with Value = std::pair<long int, unsigned int>; Key = long int; HashFunc = Hash<long int>; ExtractKey = Select1st<std::pair<long int, unsigned int> >; EqualKey = std::equal_to<long int>; HashTableIterator<Value, Key, HashFunc, ExtractKey, EqualKey>::pointer = std::pair<long int, unsigned int>*; HashTableIterator<Value, Key, HashFunc, ExtractKey, EqualKey>::value_type = std::pair<long int, unsigned int>]’
unitig_graph.cpp:363:76:   required from here
hash_table.h:50:7: warning: ignoring packed attribute because of unpacked non-POD field ‘std::pair<long int, unsigned int> HashTableNode<std::pair<long int, unsigned int> >::value’ [enabled by default]
     T value;
       ^
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c sequence_manager.cpp -o sequence_manager.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c local_assembler.cpp -o local_assembler.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c city.cpp -o city.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c lib_idba/contig_graph.cpp -o lib_idba/contig_graph.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c lib_idba/contig_graph_branch_group.cpp -o lib_idba/contig_graph_branch_group.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c lib_idba/contig_info.cpp -o lib_idba/contig_info.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c lib_idba/hash_graph.cpp -o lib_idba/hash_graph.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c lib_idba/sequence.cpp -o lib_idba/sequence.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt asm_core.cpp assembler.cpp local_assemble.cpp iterate_edges.cpp lib_idba/contig_graph.o lib_idba/contig_graph_branch_group.o lib_idba/contig_info.o lib_idba/hash_graph.o lib_idba/sequence.o succinct_dbg.o assembly_algorithms.o branch_group.o options_description.o unitig_graph.o sequence_manager.o local_assembler.o city.o -lm -lz -lpthread -o megahit_asm_core
In file included from hash_map.h:17:0,
                 from unitig_graph.h:30,
                 from assembler.cpp:34:
hash_table.h: In instantiation of ‘struct HashTableNode<std::pair<long int, unsigned int> >’:
hash_table.h:599:39:   required from ‘void HashTable<Value, Key, HashFunc, ExtractKey, EqualKey>::clear() [with Value = std::pair<long int, unsigned int>; Key = long int; HashFunc = Hash<long int>; ExtractKey = Select1st<std::pair<long int, unsigned int> >; EqualKey = std::equal_to<long int>]’
hash_table.h:277:15:   required from ‘HashTable<Value, Key, HashFunc, ExtractKey, EqualKey>::~HashTable() [with Value = std::pair<long int, unsigned int>; Key = long int; HashFunc = Hash<long int>; ExtractKey = Select1st<std::pair<long int, unsigned int> >; EqualKey = std::equal_to<long int>]’
hash_map.h:32:7:   required from here
hash_table.h:50:7: warning: ignoring packed attribute because of unpacked non-POD field ‘std::pair<long int, unsigned int> HashTableNode<std::pair<long int, unsigned int> >::value’ [enabled by default]
     T value;
       ^
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c cx1_kmer_count.cpp -o cx1_kmer_count.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c cx1_read2sdbg_s1.cpp -o cx1_read2sdbg_s1.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c cx1_read2sdbg_s2.cpp -o cx1_read2sdbg_s2.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt -c cx1_seq2sdbg.cpp -o cx1_seq2sdbg.o
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt sdbg_builder.cpp cx1_kmer_count.o options_description.o cx1_read2sdbg_s1.o cx1_read2sdbg_s2.o cx1_seq2sdbg.o sequence_manager.o -lm -lz -lpthread -o megahit_sdbg_build
g++ -O2 -Wall -Wno-unused-function -Wno-array-bounds -D__STDC_FORMAT_MACROS -funroll-loops -fprefetch-loop-arrays -fopenmp -I. -std=c++0x -static-libgcc -static-libstdc++ -mpopcnt tools/toolkit.cpp tools/contigs_to_fastg.cpp tools/read_stat.cpp tools/trim_low_qual_tail.cpp tools/filter_by_len.cpp -lm -lz -lpthread -o megahit_toolkit
chmod +x ./megahit

This assembly will take about 15 minutes and will save the assembly to a folder names "megahit_assembly". You can read about the parameters to this program, such as --memory that specifies the maximum memory that can be used on the megahit software repo, https://github.com/voutcn/megahit.


In [31]:
!megahit/megahit --memory 10e9 -l 250 --k-max 81 -r SRR172903.qc.fastq --cpu-only -o megahit_assembly


MEGAHIT 0.3.0-beta3
--- [Fri Jun 26 14:13:38 2015] Start assembly. Number of CPU threads 4 ---
--- [Fri Jun 26 14:13:38 2015] k list: 21,41,61,81 ---
--- [LIB INFO] Single-end reads: SRR172903.qc.fastq
--- [Fri Jun 26 14:13:38 2015] Extracting solid (k+1)-mers for k = 21 ---
--- [Fri Jun 26 14:13:46 2015] Building graph for k = 21 ---
--- [Fri Jun 26 14:13:56 2015] Assembling contigs from SdBG for k = 21 ---
--- [Fri Jun 26 14:14:12 2015] Local assembling k = 21 ---
--- [Fri Jun 26 14:14:14 2015] Extracting iterative edges from k = 21 to 41 ---
--- [Fri Jun 26 14:14:17 2015] Building graph for k = 41 ---
--- [Fri Jun 26 14:14:23 2015] Assembling contigs from SdBG for k = 41 ---
--- [Fri Jun 26 14:14:38 2015] Local assembling k = 41 ---
--- [Fri Jun 26 14:14:39 2015] Extracting iterative edges from k = 41 to 61 ---
--- [Fri Jun 26 14:14:41 2015] Building graph for k = 61 ---
--- [Fri Jun 26 14:14:45 2015] Assembling contigs from SdBG for k = 61 ---
--- [Fri Jun 26 14:14:56 2015] Local assembling k = 61 ---
--- [Fri Jun 26 14:14:57 2015] Extracting iterative edges from k = 61 to 81 ---
--- [Fri Jun 26 14:14:57 2015] Merging to output final contigs ---
--- [STAT] 11345 contigs, total 5254736 bp, min 200 bp, max 8884 bp, avg 463.18 bp, N50 525 bp
--- [Fri Jun 26 14:14:57 2015] ALL DONE. Time elapsed: 79.401388 seconds ---

To take a look at the assembly, let's run the khmer assembly summary program on it, the final contigs are in megahit_assembly/final.contigs.fa. Let's get statistics on all contigs greater than or equal to 200 bp.


In [32]:
!python khmer/sandbox/assemstats3.py 200 megahit_assembly/final.contigs.fa


** cutoff: 200
N	sum	max	filename
11345	5254736	8884	megahit_assembly/final.contigs.fa

9. Estimating abundances of contigs

Once the assembly is finished, you have a set of reference contigs which you can now estimate the abundance of the metagenome. The approach for doing so is identical to that shown above where you use reference genomes.

This will take about 20 minutes.


In [33]:
!bash bowtie.sh megahit_assembly/final.contigs.fa SRR172903.qc.fastq


Reference is megahit_assembly/final.contigs.fa
Reads are SRR172903.qc.fastq
Settings:
  Output files: "megahit_assembly/final.contigs.fa-bowtie-index.*.bt2"
  Line rate: 6 (line is 64 bytes)
  Lines per side: 1 (side is 64 bytes)
  Offset rate: 4 (one in 16)
  FTable chars: 10
  Strings: unpacked
  Max bucket size: default
  Max bucket size, sqrt multiplier: default
  Max bucket size, len divisor: 4
  Difference-cover sample period: 1024
  Endianness: little
  Actual local endianness: little
  Sanity checking: disabled
  Assertions: disabled
  Random seed: 0
  Sizeofs: void*:8, int:4, long:8, size_t:8
Input files DNA, FASTA:
  megahit_assembly/final.contigs.fa
Building a SMALL index
Reading reference sizes
  Time reading reference sizes: 00:00:00
Calculating joined length
Writing header
Reserving space for joined string
Joining reference sequences
  Time to join reference sequences: 00:00:00
bmax according to bmaxDivN setting: 1313684
Using parameters --bmax 985263 --dcv 1024
  Doing ahead-of-time memory usage test
  Passed!  Constructing with these parameters: --bmax 985263 --dcv 1024
Constructing suffix-array element generator
Building DifferenceCoverSample
  Building sPrime
  Building sPrimeOrder
  V-Sorting samples
  V-Sorting samples time: 00:00:00
  Allocating rank array
  Ranking v-sort output
  Ranking v-sort output time: 00:00:00
  Invoking Larsson-Sadakane on ranks
  Invoking Larsson-Sadakane on ranks time: 00:00:00
  Sanity-checking and returning
Building samples
Reserving space for 12 sample suffixes
Generating random suffixes
QSorting 12 sample offsets, eliminating duplicates
QSorting sample offsets, eliminating duplicates time: 00:00:00
Multikey QSorting 12 samples
  (Using difference cover)
  Multikey QSorting samples time: 00:00:00
Calculating bucket sizes
  Binary sorting into buckets
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  Binary sorting into buckets time: 00:00:00
Splitting and merging
  Splitting and merging time: 00:00:00
Split 2, merged 5; iterating...
  Binary sorting into buckets
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  Binary sorting into buckets time: 00:00:01
Splitting and merging
  Splitting and merging time: 00:00:00
Avg bucket size: 583859 (target: 985262)
Converting suffix-array elements to index image
Allocating ftab, absorbFtab
Entering Ebwt loop
Getting block 1 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 409513
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 409514
Getting block 2 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 717594
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 717595
Getting block 3 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 275862
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 275863
Getting block 4 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 933544
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 933545
Getting block 5 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 456717
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 456718
Getting block 6 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:01
  Sorting block of length 650578
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 650579
Getting block 7 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 551983
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 551984
Getting block 8 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 736987
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 736988
Getting block 9 of 9
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 521950
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 521951
Exited Ebwt loop
fchr[A]: 0
fchr[C]: 1811471
fchr[G]: 2639376
fchr[T]: 3476168
fchr[$]: 5254736
Exiting Ebwt::buildToDisk()
Returning from initFromVector
Wrote 6549449 bytes to primary EBWT file: megahit_assembly/final.contigs.fa-bowtie-index.1.bt2
Wrote 1313692 bytes to secondary EBWT file: megahit_assembly/final.contigs.fa-bowtie-index.2.bt2
Re-opening _in1 and _in2 as input streams
Returning from Ebwt constructor
Headers:
    len: 5254736
    bwtLen: 5254737
    sz: 1313684
    bwtSz: 1313685
    lineRate: 6
    offRate: 4
    offMask: 0xfffffff0
    ftabChars: 10
    eftabLen: 20
    eftabSz: 80
    ftabLen: 1048577
    ftabSz: 4194308
    offsLen: 328422
    offsSz: 1313688
    lineSz: 64
    sideSz: 64
    sideBwtSz: 48
    sideBwtLen: 192
    numSides: 27369
    numLines: 27369
    ebwtTotLen: 1751616
    ebwtTotSz: 1751616
    color: 0
    reverse: 0
Total time for call to driver() for forward index: 00:00:02
Reading reference sizes
  Time reading reference sizes: 00:00:00
Calculating joined length
Writing header
Reserving space for joined string
Joining reference sequences
  Time to join reference sequences: 00:00:00
  Time to reverse reference sequence: 00:00:00
bmax according to bmaxDivN setting: 1313684
Using parameters --bmax 985263 --dcv 1024
  Doing ahead-of-time memory usage test
  Passed!  Constructing with these parameters: --bmax 985263 --dcv 1024
Constructing suffix-array element generator
Building DifferenceCoverSample
  Building sPrime
  Building sPrimeOrder
  V-Sorting samples
  V-Sorting samples time: 00:00:00
  Allocating rank array
  Ranking v-sort output
  Ranking v-sort output time: 00:00:00
  Invoking Larsson-Sadakane on ranks
  Invoking Larsson-Sadakane on ranks time: 00:00:00
  Sanity-checking and returning
Building samples
Reserving space for 12 sample suffixes
Generating random suffixes
QSorting 12 sample offsets, eliminating duplicates
QSorting sample offsets, eliminating duplicates time: 00:00:00
Multikey QSorting 12 samples
  (Using difference cover)
  Multikey QSorting samples time: 00:00:00
Calculating bucket sizes
  Binary sorting into buckets
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  Binary sorting into buckets time: 00:00:00
Splitting and merging
  Splitting and merging time: 00:00:00
Avg bucket size: 656841 (target: 985262)
Converting suffix-array elements to index image
Allocating ftab, absorbFtab
Entering Ebwt loop
Getting block 1 of 8
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 537964
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 537965
Getting block 2 of 8
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 965284
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 965285
Getting block 3 of 8
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 368748
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 368749
Getting block 4 of 8
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:01
  Sorting block of length 970675
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 970676
Getting block 5 of 8
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 562616
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 562617
Getting block 6 of 8
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 804480
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 804481
Getting block 7 of 8
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:00
  Sorting block of length 952899
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 952900
Getting block 8 of 8
  Reserving size (985263) for bucket
  Calculating Z arrays
  Calculating Z arrays time: 00:00:00
  Entering block accumulator loop:
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  Block accumulator loop time: 00:00:01
  Sorting block of length 92063
  (Using difference cover)
  Sorting block time: 00:00:00
Returning block of 92064
Exited Ebwt loop
fchr[A]: 0
fchr[C]: 1811471
fchr[G]: 2639376
fchr[T]: 3476168
fchr[$]: 5254736
Exiting Ebwt::buildToDisk()
Returning from initFromVector
Wrote 6549449 bytes to primary EBWT file: megahit_assembly/final.contigs.fa-bowtie-index.rev.1.bt2
Wrote 1313692 bytes to secondary EBWT file: megahit_assembly/final.contigs.fa-bowtie-index.rev.2.bt2
Re-opening _in1 and _in2 as input streams
Returning from Ebwt constructor
Headers:
    len: 5254736
    bwtLen: 5254737
    sz: 1313684
    bwtSz: 1313685
    lineRate: 6
    offRate: 4
    offMask: 0xfffffff0
    ftabChars: 10
    eftabLen: 20
    eftabSz: 80
    ftabLen: 1048577
    ftabSz: 4194308
    offsLen: 328422
    offsSz: 1313688
    lineSz: 64
    sideSz: 64
    sideBwtSz: 48
    sideBwtLen: 192
    numSides: 27369
    numLines: 27369
    ebwtTotLen: 1751616
    ebwtTotSz: 1751616
    color: 0
    reverse: 1
Total time for backward call to driver() for mirror index: 00:00:03
820417 reads; of these:
  820417 (100.00%) were unpaired; of these:
    298465 (36.38%) aligned 0 times
    513783 (62.62%) aligned exactly 1 time
    8169 (1.00%) aligned >1 times
63.62% overall alignment rate
[samopen] SAM header is present: 11345 sequences.

You can take a look at the results of the mapping much like you did above when we were mapping reads to the NCBI genome.


In [34]:
!cat reads-mapped.count.txt


521952

In [35]:
!cat reads-unmapped.count.txt


298465

In [36]:
!cat reads.by.contigs.txt


k61_2	218	15	0
k61_3	206	6	0
k61_4	256	104	0
k61_5	258	14	0
k61_6	305	21	0
k61_7	284	13	0
k61_8	256	63	0
k61_9	593	51	0
k61_10	400	36	0
k61_11	463	26	0
k61_12	270	12	0
k61_13	344	26	0
k61_14	335	31	0
k61_15	243	14	0
k61_16	481	42	0
k61_17	456	17	0
k61_18	283	15	0
k61_19	428	27	0
k61_20	763	78	0
k61_21	240	20	0
k61_22	361	25	0
k61_23	1476	157	0
k61_24	441	24	0
k61_25	1197	102	0
k61_27	354	23	0
k61_28	567	34	0
k61_29	353	17	0
k61_30	988	69	0
k61_31	294	11	0
k61_32	334	24	0
k61_33	419	26	0
k61_34	644	54	0
k61_36	767	88	0
k61_37	318	23	0
k61_38	398	22	0
k61_39	469	58	0
k61_40	229	7	0
k61_41	279	12	0
k61_42	577	47	0
k61_44	606	54	0
k61_45	455	45	0
k61_46	248	24	0
k61_47	206	10	0
k61_48	615	59	0
k61_49	976	93	0
k61_50	570	67	0
k61_52	218	6	0
k61_53	1128	128	0
k61_54	210	8	0
k61_55	225	18	0
k61_56	262	20	0
k61_58	220	24	0
k61_59	400	19	0
k61_60	519	46	0
k61_61	203	9	0
k61_62	873	120	0
k61_63	368	16	0
k61_65	208	18	0
k61_66	410	39	0
k61_67	1100	169	0
k61_68	365	13	0
k61_69	277	23	0
k61_70	472	42	0
k61_71	475	39	0
k61_72	328	16	0
k61_73	225	23	0
k61_74	682	65	0
k61_75	440	41	0
k61_76	268	17	0
k61_77	280	31	0
k61_78	225	7	0
k61_79	295	20	0
k61_80	384	41	0
k61_81	209	5	0
k61_82	247	12	0
k61_83	1478	158	0
k61_84	256	12	0
k61_85	372	40	0
k61_86	370	25	0
k61_87	368	48	0
k61_88	370	20	0
k61_89	277	13	0
k61_90	673	50	0
k61_91	633	47	0
k61_92	327	16	0
k61_93	225	9	0
k61_94	403	35	0
k61_95	237	12	0
k61_96	337	20	0
k61_97	206	5	0
k61_98	254	9	0
k61_99	310	16	0
k61_101	715	65	0
k61_102	288	21	0
k61_103	590	54	0
k61_104	573	32	0
k61_105	2060	222	0
k61_106	454	21	0
k61_107	297	27	0
k61_108	755	63	0
k61_110	352	27	0
k61_111	294	23	0
k61_112	288	27	0
k61_113	230	16	0
k61_114	257	15	0
k61_115	748	58	0
k61_117	420	16	0
k61_118	370	26	0
k61_119	309	16	0
k61_120	858	88	0
k61_122	273	12	0
k61_123	278	15	0
k61_124	281	25	0
k61_125	353	18	0
k61_126	329	28	0
k61_127	247	14	0
k61_128	217	18	0
k61_129	543	57	0
k61_130	486	62	0
k61_131	299	23	0
k61_132	279	6	0
k61_133	687	69	0
k61_135	208	15	0
k61_139	212	7	0
k61_141	216	15	0
k61_142	318	15	0
k61_143	815	68	0
k61_144	286	19	0
k61_145	537	54	0
k61_146	462	36	0
k61_147	276	18	0
k61_148	479	168	0
k61_149	232	11	0
k61_150	895	95	0
k61_151	332	14	0
k61_152	236	9	0
k61_153	303	13	0
k61_154	494	51	0
k61_155	486	44	0
k61_156	807	72	0
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*	0	0	298465

10. Annotating the assembled contigs

Sequencing is often used to determine "who" and/or "what" is in your sample. In our case, we know that the HMP mock community should orignate from a set of genomes (which we actually downlaoded above). One of the most popular tools of comparing an unknown sequence to a known reference is The Basic Local Alignment Search Tool (or BLAST). To identify the origin of our contigs, we will align assembled contigs to the genomes used in the HMP mock community.

The first thing we will do is download the BLAST software. Given the increasing volume of sequencing datasets, one may also consider new tools for more efficient annotation are now available such as Diamond (https://github.com/bbuchfink/diamond/, http://dx.doi.org/10.1038/nmeth.3176).


In [40]:
!wget ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.30/ncbi-blast-2.2.30+-x64-linux.tar.gz
!tar -xvf ncbi-blast-2.2.30+-x64-linux.tar.gz


--2015-06-26 14:20:56--  ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.30/ncbi-blast-2.2.30+-x64-linux.tar.gz
           => ‘ncbi-blast-2.2.30+-x64-linux.tar.gz’
Resolving ftp.ncbi.nlm.nih.gov (ftp.ncbi.nlm.nih.gov)... 130.14.250.12, 2607:f220:41e:250::11
Connecting to ftp.ncbi.nlm.nih.gov (ftp.ncbi.nlm.nih.gov)|130.14.250.12|:21... connected.
Logging in as anonymous ... Logged in!
==> SYST ... done.    ==> PWD ... done.
==> TYPE I ... done.  ==> CWD (1) /blast/executables/blast+/2.2.30 ... done.
==> SIZE ncbi-blast-2.2.30+-x64-linux.tar.gz ... 172818286
==> PASV ... done.    ==> RETR ncbi-blast-2.2.30+-x64-linux.tar.gz ... done.
Length: 172818286 (165M) (unauthoritative)

100%[======================================>] 172,818,286  164MB/s   in 1.0s   

2015-06-26 14:20:57 (164 MB/s) - ‘ncbi-blast-2.2.30+-x64-linux.tar.gz’ saved [172818286]

ncbi-blast-2.2.30+/
ncbi-blast-2.2.30+/LICENSE
ncbi-blast-2.2.30+/ChangeLog
ncbi-blast-2.2.30+/ncbi_package_info
ncbi-blast-2.2.30+/doc/
ncbi-blast-2.2.30+/doc/README.txt
ncbi-blast-2.2.30+/README
ncbi-blast-2.2.30+/bin/
ncbi-blast-2.2.30+/bin/tblastn
ncbi-blast-2.2.30+/bin/blastdbcheck
ncbi-blast-2.2.30+/bin/blast_formatter
ncbi-blast-2.2.30+/bin/deltablast
ncbi-blast-2.2.30+/bin/tblastx
ncbi-blast-2.2.30+/bin/blastp
ncbi-blast-2.2.30+/bin/makeblastdb
ncbi-blast-2.2.30+/bin/blastn
ncbi-blast-2.2.30+/bin/windowmasker
ncbi-blast-2.2.30+/bin/blastdbcmd
ncbi-blast-2.2.30+/bin/makembindex
ncbi-blast-2.2.30+/bin/makeprofiledb
ncbi-blast-2.2.30+/bin/update_blastdb.pl
ncbi-blast-2.2.30+/bin/blastdb_aliastool
ncbi-blast-2.2.30+/bin/dustmasker
ncbi-blast-2.2.30+/bin/psiblast
ncbi-blast-2.2.30+/bin/rpsblast
ncbi-blast-2.2.30+/bin/rpstblastn
ncbi-blast-2.2.30+/bin/convert2blastmask
ncbi-blast-2.2.30+/bin/blastx
ncbi-blast-2.2.30+/bin/segmasker
ncbi-blast-2.2.30+/bin/legacy_blast.pl

Now, we will make a searchable database for the BLAST software. First, we'll concatenate all the genomes in the genomes directory to one file.


In [41]:
!cat genomes/*fa >> all-genomes.fa
!ncbi-blast-2.2.30+/bin/makeblastdb -in all-genomes.fa -dbtype nucl -out all-genomes
!ncbi-blast-2.2.30+/bin/blastn -db all-genomes -query megahit_assembly/final.contigs.fa -outfmt 6 -out contigs.x.all-genomes.blastnout



Building a new DB, current time: 06/26/2015 14:21:06
New DB name:   all-genomes
New DB title:  all-genomes.fa
Sequence type: Nucleotide
Keep Linkouts: T
Keep MBits: T
Maximum file size: 1000000000B
Adding sequences from FASTA; added 60 sequences in 1.84624 seconds.

The above command aligns each query (each sequence in the assembled final.contings.fa file) with each sequence (e.g., genome in all-genomes.fa). The -outfmt tells the program to save the results in a tab-delimited format in the -out file contigs.x.all-genomes.blastnout.

Let's take a look at the first 10 lines of that file. You'll see the query (contig) and the hit (genome) followed by the percent identity, the length of alignment, mismatch counts, gap open counts, query start position, query end position, subject start position, subject end position, E-value, and bit score.


In [42]:
!head -n 10 contigs.x.all-genomes.blastnout


k61_2	gi|27466918|ref|NC_004461.1|	100.00	218	0	0	1	218	1986675	1986892	5e-112	  403
k61_2	gi|27466918|ref|NC_004461.1|	100.00	218	0	0	1	218	1986675	1986892	5e-112	  403
k61_3	gi|24378532|ref|NC_004350.1|	100.00	206	0	0	1	206	1898492	1898697	2e-105	  381
k61_3	gi|24378532|ref|NC_004350.1|	100.00	206	0	0	1	206	1898492	1898697	2e-105	  381
k61_4	gi|32470532|ref|NC_005004.1|	95.31	256	12	0	1	256	10969	10714	4e-113	  407
k61_4	gi|32470532|ref|NC_005004.1|	95.31	256	12	0	1	256	10969	10714	4e-113	  407
k61_4	gi|32470555|ref|NC_005005.1|	92.58	256	19	0	1	256	10016	10271	2e-101	  368
k61_4	gi|32470555|ref|NC_005005.1|	92.58	256	19	0	1	256	10016	10271	2e-101	  368
k61_5	gi|24378532|ref|NC_004350.1|	100.00	258	0	0	1	258	928208	928465	3e-134	  477
k61_5	gi|24378532|ref|NC_004350.1|	100.00	258	0	0	1	258	928208	928465	3e-134	  477

Depending on your scientific question, it may be of more interest to have open reading frames (ORFs) annotated rather than contig sequences. In this case, there exist multiple ORF callers (e.g., FragGeneScan, http://nar.oxfordjournals.org/content/early/2010/08/29/nar.gkq747.abstract and Metagene, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1636498/) that can be used. We can call ORFs from our contigs using FragGeneScan. We will download, install, and then call ORFs from our contigs as follows:


In [43]:
!wget http://downloads.sourceforge.net/project/fraggenescan/FragGeneScan1.19.tar.gz
!tar -xvf FragGeneScan1.19.tar.gz


--2015-06-26 14:21:15--  http://downloads.sourceforge.net/project/fraggenescan/FragGeneScan1.19.tar.gz
Resolving downloads.sourceforge.net (downloads.sourceforge.net)... 216.34.181.59
Connecting to downloads.sourceforge.net (downloads.sourceforge.net)|216.34.181.59|:80... connected.
HTTP request sent, awaiting response... 302 Found
Location: http://iweb.dl.sourceforge.net/project/fraggenescan/FragGeneScan1.19.tar.gz [following]
--2015-06-26 14:21:15--  http://iweb.dl.sourceforge.net/project/fraggenescan/FragGeneScan1.19.tar.gz
Resolving iweb.dl.sourceforge.net (iweb.dl.sourceforge.net)... 70.38.0.134, 2607:f748:10:12::5f:2
Connecting to iweb.dl.sourceforge.net (iweb.dl.sourceforge.net)|70.38.0.134|:80... connected.
HTTP request sent, awaiting response... 200 OK
Length: 11371331 (11M) [application/x-gzip]
Saving to: ‘FragGeneScan1.19.tar.gz’

100%[======================================>] 11,371,331   674KB/s   in 15s    

2015-06-26 14:21:30 (738 KB/s) - ‘FragGeneScan1.19.tar.gz’ saved [11371331/11371331]

FragGeneScan1.19/
FragGeneScan1.19/post_process.pl
FragGeneScan1.19/run_FragGeneScan.pl
FragGeneScan1.19/hmm.h
FragGeneScan1.19/README
FragGeneScan1.19/hmm_lib.c
FragGeneScan1.19/Makefile
FragGeneScan1.19/run_hmm.c
FragGeneScan1.19/example/
FragGeneScan1.19/example/NC_000913.test.454.5.gff
FragGeneScan1.19/example/NC_000913.test.ffn
FragGeneScan1.19/example/NC_000913.test.out
FragGeneScan1.19/example/NC_000913.test.gff
FragGeneScan1.19/example/NC_000913-454.test.gff
FragGeneScan1.19/example/NC_000913.test.454.5.out
FragGeneScan1.19/example/NC_000913-454.test.out
FragGeneScan1.19/example/NC_000913.test.454.5.faa
FragGeneScan1.19/example/NC_000913-454.fna
FragGeneScan1.19/example/NC_000913.fna
FragGeneScan1.19/example/NC_000913.test.454.5.ffn
FragGeneScan1.19/example/NC_000913-454.test.ffn
FragGeneScan1.19/example/NC_000913.test.faa
FragGeneScan1.19/example/NC_000913-454.test.faa
FragGeneScan1.19/util_lib.h
FragGeneScan1.19/util_lib.c
FragGeneScan1.19/FGS_gff.py
FragGeneScan1.19/train/
FragGeneScan1.19/train/illumina_10
FragGeneScan1.19/train/454_5
FragGeneScan1.19/train/pwm
FragGeneScan1.19/train/454_30
FragGeneScan1.19/train/rgene
FragGeneScan1.19/train/stop1
FragGeneScan1.19/train/noncoding
FragGeneScan1.19/train/complete
FragGeneScan1.19/train/start
FragGeneScan1.19/train/454_10
FragGeneScan1.19/train/start1
FragGeneScan1.19/train/illumina_5
FragGeneScan1.19/train/sanger_10
FragGeneScan1.19/train/gene
FragGeneScan1.19/train/illumina_1
FragGeneScan1.19/train/stop
FragGeneScan1.19/train/sanger_5

In [44]:
!bash fraggenescan-install.sh


rm -rf *.o FragGeneScan* *~
gcc    -c -o util_lib.o util_lib.c
gcc    -c -o hmm_lib.o hmm_lib.c
gcc    -c -o run_hmm.o run_hmm.c
gcc  -O3 -o FragGeneScan util_lib.o hmm_lib.o run_hmm.o  -lm -lpthread

We will run FragGeneScan on the assembled contigs, assuming that it fits the training profile of a "complete" genome sequence (in their documentaion, this equals complete genomic sequences or short sequence reads without sequencing error).


In [45]:
!FragGeneScan1.19/FragGeneScan -s megahit_assembly/final.contigs.fa -o final.contigs.orfs.fa -w 1 -t complete


no. of seqs: 11345

The ORFs called are contained as FASTA files in final.contigs.orfs.fa.faa (amino acids) and final.contigs.orfs.fa.ffn (nucleotides). You can annotate these against a database of your choice just as described for the contigs above.

11. Going forward

Now you have all the information you need to produce the following information:

  • Sequence Abundance Information: Sequence (e.g., contig) and abundance (e.g., number of mapped reads)
  • Sequence Annotation Information: Sequence (eg., contig) and NCBI genome

You'll note that this is similar to 16S rRNA amplicon analysis where you'd have an OTU abundance table and OTU best hit annotations. For metagenomic analysis, this information takes you into further analysis and visualization packages like PhyloSeq in R (http://joey711.github.io/phyloseq/).

Once you run through this tutorial on this workbook, a good exercise would be to try to run the assembly outside the IPython Notebook environment. To do so, you can log into your EC2 instance, navigate to the directory where this data is stored (cd /mnt/frontiers-review-2015), and you could run every command in this notebook on the command line (with the exception of the "!" at the beginning of each command in the notebook. Also, note that you will not have to reinstall the software.


In [ ]: