Mapping to Reference

Motivation

Mapping to a reference genome is a vital step to generate counts and do differential gene expression thereafter. For RNA-Seq data it is important to choose an aligner which is splice-aware.

Aims

In this part you will learn to:

• align RNA-Seq reads to a reference genome
• calculate the mapping rate

You will use the following software:

• TopHat2 v2.0.11: http://ccb.jhu.edu/software/tophat/index.shtml
• Bowtie2 v2.2.0: http://bowtie-bio.sourceforge.net/bowtie2/index.shtml

The data set you'll be using is downloaded from ENA (http://www.ebi.ac.uk/ena/data/view/SRP019027). The reads belong to sample SRR769316. The data set is tailored with respect to the time allocated for the exercise.

Indexing

Go to the right folder/directory:

cd $HOME/i2rda_data/Mapping_to_Reference

Index the reference genome using one of the Bowtie2:

cd Reference
bowtie2-build mm10_chr19-1-20000000.fa mm10_chr19-1-20000000

Note the "fa" extension for the reference this is due to a preference of tophat which we'll be using below.

Run the alignment using TopHat2:

cd ..
tophat -o tophat2 --no-mixed --rg-id Lane-1 --rg-sample sample1 --rg-center XYZ --rg-platform Illumina -G Reference/mm10_chr19-1-20000000_Ensembl.gtf Reference/mm10_chr19-1-20000000 $HOME/i2rda_data/Mapping_to_Reference/Read_1.fastq.gz $HOME/i2rda_data/Mapping_to_Reference/Read_2.fastq.gz

where:

• --no-mixed: For paired reads, only report read alignments if both reads in a pair can be mapped
• --rg-id: Read group ID
• --rg-sample: Sample ID
• --rg-center: Sequencing Centre name
• --rg-platform: Sequencing platform descriptor
• -G: Supply TopHat with a set of gene model annotations and/or known transcripts, as a GTF 2.2 or GFF3 formatted file.
• -o: Output directory

Check the output of TopHat2:

cd tophat2
ls

Get the mapping rate:

cat align_summary.txt

Get the number of reads mapped. Run the alignment of filtered data using TopHat2

cd ..
tophat2 -o tophat2_with_filtered_data --no-mixed \
--rg-id Lane-1 --rg-sample sample1 --rg-center XYZ --rg-platform Illumina \
-G Reference/mm10_chr19-1-20000000_Ensembl.gtf Reference/mm10_chr19-1-20000000 \
$HOME/i2rda_data/Mapping_to_Reference/Read_1_q30l50.fastq.gz \
$HOME/i2rda_data/Mapping_to_Reference/Read_2_q30l50.fastq.gz

Check the output of TopHat2:

cd tophat2_with_filtered_data
ls

Get the mapping rate:

cat align_summary.txt

Get the number of reads mapped.

• What difference does using the filtered data make?