Rf: s2

2017-04-08T22:25:55Z

Rf: Rf moved page Estimating Gen Count Exercise to Estimating Gene Count Exercise

2017-04-08T22:22:01Z

Rf moved page Estimating Gen Count Exercise to Estimating Gene Count Exercise

New page

#REDIRECT [[Estimating Gene Count Exercise]]

← Older revision		Revision as of 22:25, 8 April 2017
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−	~~#REDIRECT~~ [~~[Estimating Gene~~ Count ~~Exercise]]~~	+	= Aims =
		+
		+	In this part you will learn to:
		+	* generate count tables
		+
		+	HTSeq v0.6.1p1: http://www-huber.embl.de/users/anders/HTSeq/doc/count.html
		+	The data set you'll be using is downloaded from ENA (http://www.ebi.ac.uk/ena/data/view/SRP019027). The reads belong to
		+	samples SRR769314 and SRR769316. The data set is tailored with respect to the time allocated for the workshop. Reads were alig
		+	ned to the first 20 Mb of chromosome 19 of the mouse reference genome (GRCm38/mm10) using TopHat and duplicates marked
		+	using Picard MarkDuplicates.
		+	You will use the following files:
		+	SRR769314_duplicates_marked.bam: aligned reads
		+	SRR769316_duplicates_marked.bam: aligned reads
		+	mm10_chr19-1-20000000_Ensembl.gtf: Ensembl mouse gene models
		+
		+	Type text like this in the terminal at the $ command prompt, then press the
		+	[Enter] key to run the command.
		+
		+	= Data =
		+	The data is available in the directory <code>07_Estimating_gene_count</code>:
		+
		+	cd /home/training/Data/07_Estimating_gene_count
		+
		+	= Generate count tables =
		+	Generate a count table for sample SRR769314 using HTSeq's <code>htseq-count</code>:
		+
		+	htseq-count -f bam -r pos -t exon \
		+	-s no -m intersection-nonempty \
		+	SRR769314_duplicates_marked.bam \
		+	Reference/mm10_chr19-1-20000000_Ensembl.gtf \
		+	> SRR769314_duplicates_marked_htseq.count
		+
		+	<ins>where</ins>:
		+	* <code>-f</code>: format of the input data (default: sam).
		+	* <code>-r</code>: for paired-end data, the alignment have to be sorted either by read name or by alignment position (default: name).
		+	* <code>-t</code>: feature type (3rd column in GFF/GTF file) to be used, all features of other type are ignored (default: exon).
		+	* <code>-s</code>: whether the data is from a strand-specific assay (default: yes).
		+	* <code>-m</code>: mode to handle reads overlapping more than one feature (default: union).
		+
		+	Repeat for sample SRR769316:
		+
		+	htseq-count -f bam -r pos -t exon \
		+	-s no -m intersection-nonempty \
		+	SRR769316_duplicates_marked.bam \
		+	Reference/mm10_chr19-1-20000000_Ensembl.gtf \
		+	> SRR769316_duplicates_marked_htseq.count
		+
		+	Have a look at the start and end of the results files:
		+
		+	head SRR769314_duplicates_marked_htseq.count
		+	tail SRR769314_duplicates_marked_htseq.count
		+	head SRR769316_duplicates_marked_htseq.count
		+	tail SRR769316_duplicates_marked_htseq.count
		+
		+	Count the proportion of reads that fall in annotated genes, using a small AWK script:
		+
		+	awk '{if(/^__/){excluded+=$2}else{included+=$2}} \
		+	END{printf "%s,%s,%.2f%%\n", included, excluded,\
		+	included/(included+excluded)*100}' \
		+	SRR769314_duplicates_marked_htseq.count
		+	awk '{if(/^__/){excluded+=$2}else{included+=$2}} \
		+	END{printf "%s,%s,%.2f%%\n", included, excluded, \
		+	included/(included+excluded)*100}' \
		+	SRR769316_duplicates_marked_htseq.count
		+
		+	which means:
		+	for each line in the count file:
		+	if the line contains the pattern __ at the start of the line
		+	increase the value of the variable excluded with the value in the second field of the line
		+	else increase the value of the variable included with the value in the second field of the line
		+	then:
		+	print the value of included, excluded and included/(included+excluded)*100, as a string, a string and a floating point number with two decimal place, respectively, followed by a line break
		+
		+	* What proportion of read are excluded from further analysis?
		+	* What is the main reason for reads being excluded?
		+
		+	Run HTSeq htseq-count again changing the mode from intersection-nonempty to union.
		+	* What difference does this make?

Estimating Gen Count Exercise - Revision history

Rf: s2

Rf: Rf moved page Estimating Gen Count Exercise to Estimating Gene Count Exercise