I2rda m2rx
Aims
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.
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.
Change directory:
cd /home/training/Data/04_Mapping_to_a_reference_genome
Index the reference genome using Bowtie2:
cd Reference bowtie2-build mm10_chr19-1-20000000.fasta mm10_chr19-1-20000000
Run the alignment using TopHat2:
cd .. tophat2 -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/training/Data/03_Quality_control_and_data_preprocessing/Read_1.fastq \ /home/training/Data/03_Quality_control_and_data_preprocessing/Read_2.fastq
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/training/Data/03_Quality_control_and_data_preprocessing/Read_1_q30l50.fastq \ /home/training/Data/03_Quality_control_and_data_preprocessing/Read_2_q30l50.fastq
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?