Rf at 14:52, 7 May 2017

2017-05-07T14:52:11Z

Rf: Rf moved page Mapping to Reference to I2rda m2rx

2017-05-04T12:52:56Z

Rf moved page Mapping to Reference to I2rda m2rx

New page

#REDIRECT [[I2rda m2rx]]

← Older revision		Revision as of 14:52, 7 May 2017
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−	~~#REDIRECT [[I2rda m2rx]]~~	+	= 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?

Mapping to Reference - Revision history

Rf at 14:52, 7 May 2017

Rf: Rf moved page Mapping to Reference to I2rda m2rx