Next Generation Sequencing Pipeline

This is a series of steps in a generic NGS pipeline to learn how to do NGS analysis.

Author: R. Burke Squires

Outline

Sequencers and sequencing technology
Evaluating sequencing data
Pre-processing your data
Assembly
- Denovo assembly (without a reference sequecne)
- Assembly (with a reference sequence)

Investigate your sequencing data

First, where is your sequencing data?



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seq_file = ""

First, lets run FASTQC to examine the sequence quality of your data



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!fastqc seq_file









    



/bin/sh: fastqc: command not found

Pre-processing data

Trimmomatic



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TrimmomaticPE: Started with arguments: -phred33 Para6-10_S2_L001_R1_001.fastq Para6-10_S2_L001_R2_001.fastq output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Multiple cores found: Using 8 threads
Oct 10, 2014 2:06:15 PM org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer makeIlluminaClippingTrimmer
SEVERE: null
java.io.FileNotFoundException: /Users/squiresrb/iPython-Notebooks/NGS/TruSeq3-PE.fa (No such file or directory)
	at java.io.FileInputStream.open(Native Method)
	at java.io.FileInputStream.<init>(FileInputStream.java:120)
	at org.usadellab.trimmomatic.fasta.FastaParser.parse(FastaParser.java:54)
	at org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer.loadSequences(IlluminaClippingTrimmer.java:107)
	at org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer.makeIlluminaClippingTrimmer(IlluminaClippingTrimmer.java:70)
	at org.usadellab.trimmomatic.trim.TrimmerFactory.makeTrimmer(TrimmerFactory.java:27)
	at org.usadellab.trimmomatic.TrimmomaticPE.run(TrimmomaticPE.java:495)
	at org.usadellab.trimmomatic.Trimmomatic.main(Trimmomatic.java:35)
Exception in thread "main" java.io.FileNotFoundException: Para6-10_S2_L001_R1_001.fastq (No such file or directory)
	at java.io.FileInputStream.open(Native Method)
	at java.io.FileInputStream.<init>(FileInputStream.java:120)
	at org.usadellab.trimmomatic.fastq.FastqParser.parse(FastqParser.java:127)
	at org.usadellab.trimmomatic.TrimmomaticPE.process(TrimmomaticPE.java:251)
	at org.usadellab.trimmomatic.TrimmomaticPE.run(TrimmomaticPE.java:498)
	at org.usadellab.trimmomatic.Trimmomatic.main(Trimmomatic.java:35)



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