RNA-seq workshop ALIGNMENT

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Presentation transcript:

RNA-seq workshop ALIGNMENT Erin Osborne Nishimura http:onish.web.unc/HTSF-workshop-2015

Alignment with Tophat2

There are many alignment tools available Nuno A. Fonseca et al. Bioinformatics 2012;28:3169-3177 © The Author 2012. Published by Oxford University Press.

Which aligner is best? What type of data do you have? What is your research question?

Methods of alignment Splice awareness: What will be matched first? Splice unaware (Bowtie, BWA) Faster Splice aware (Tophat, MapSplice, SpliceMap) Slower Yields more information on splice junctions What will be matched first? Whole genome? Known transcriptome? A short segment of each read first?

Why tophat? Popular Splice aware de novo or sequenced genome modes Transcriptome or whole genome assembly Lots of options for customization Drawbacks Lots of parameters to set & optimize

Tophat2 – how does it work? Kim et al., 2013 Genome Biology http://www.genomebiology.com/2013/14/4/r36

Tophat2 versus Tophat1

The good news… … choice of aligner does not have a major impact on genes identified as differentially expressed, compared to other choices. Fonseca, 2014

Switch to Tophat2 Tutorial https://github.com/erinosb/HTSF_workshop/blob/master/01_RNAseq_alignment.md

Generating genome browser tracks

Ah, those beautiful browser tracks… Brooks and Yang et al., 2011, Genome Research

Today’s simple analysis pipeline .fastq file trimmomatic/bbduk.sh _trim.fastq file TOPHAT2 .bam/.sam file HTseq bedtools genomecov counts.txt file .bg file bedGraphToBigWig DESeq2/R .bw file Differentially Abundant genes IGV/UCSC Pretty browser shots

I have included an example script for Script05_makeBrowserTracks.sh Requires bedGraphToBigWig Requires bedtools Performs Normalization Normalize to read depth One option Scale = (#bps in genome) (#bp per read) x (# mapped reads)

Two most common platforms IGV https://www.broadinstitute.org/igv/ Locally installed UCSC Genome Browser http://genome.ucsc.edu/ Upload required

Visual inspection of each normalized replicate is critical… http://genome.ucsc.edu/cgi-bin/hgTracks?hgS_doOtherUser=submit&hgS_otherUserName=Erin%20Osborne&hgS_otherUserSessionName=hg19_female_v_male_demo http://bit.ly/1SJrQcq