1. RNA-seq workshop ALIGNMENT
Erin Osborne Nishimura

2. Alignment with Tophat2

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

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

5. 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?

6. 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

7. Tophat2 – how does it work?
Kim et al., 2013 Genome Biology

8. Tophat2 versus Tophat1

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

10. Switch to Tophat2 Tutorial

11. Generating genome browser tracks

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

13. 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

14. 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)

15. Two most common platforms
IGV
Locally installed
UCSC Genome Browser
Upload required

16. Visual inspection of each normalized replicate is critical…