Difference between revisions of "Mapping to Reference Exercise"
(Created page with "= 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 aligne...") |
|||
| Line 1: | Line 1: | ||
| + | = 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 = | = Aims = | ||
| − | |||
| − | |||
In this part you will learn to: | In this part you will learn to: | ||
| Line 13: | Line 15: | ||
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. | 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 / | + | cd $HOME/i2rda_data/Mapping_to_Reference |
| − | Index the reference genome using Bowtie2: | + | Index the reference genome using one of the Bowtie2: |
cd Reference | cd Reference | ||
| Line 28: | Line 32: | ||
--rg-id Lane-1 --rg-sample sample1 --rg-center XYZ --rg-platform Illumina \ | --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 \ | -G Reference/mm10_chr19-1-20000000_Ensembl.gtf Reference/mm10_chr19-1-20000000 \ | ||
| − | / | + | $HOME/i2rda_data/Mapping_to_Reference/Read_1.fastq \ |
| − | / | + | $HOME/i2rda_data/Mapping_to_Reference/Read_2.fastq |
where: | where: | ||
| Line 56: | Line 60: | ||
--rg-id Lane-1 --rg-sample sample1 --rg-center XYZ --rg-platform Illumina \ | --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 \ | -G Reference/mm10_chr19-1-20000000_Ensembl.gtf Reference/mm10_chr19-1-20000000 \ | ||
| − | / | + | $HOME/i2rda_data/Mapping_to_Reference/Read_1_q30l50.fastq \ |
| − | / | + | $HOME/i2rda_data/Mapping_to_Reference/Read_2_q30l50.fastq |
Check the output of TopHat2: | Check the output of TopHat2: | ||
Revision as of 16:40, 6 May 2017
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.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/i2rda_data/Mapping_to_Reference/Read_1.fastq \ $HOME/i2rda_data/Mapping_to_Reference/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/i2rda_data/Mapping_to_Reference/Read_1_q30l50.fastq \ $HOME/i2rda_data/Mapping_to_Reference/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?
