Estimating Gene Count Exercise

Aims

In this part you will learn to:

• generate count tables

The tool you will use is:

• 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 exercise. Reads were aligned to the first 20 Mb of chromosome 19 of the mouse reference genome (GRCm38/mm10) using TopHat and duplicates marked using Picard- tools' 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

Data

The data is available in the directory 07_Estimating_gene_count:

cd /home/training/Data/07_Estimating_gene_count

Generate count tables

Generate a count table for sample SRR769314 using HTSeq's htseq-count:

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

where:

• -f: format of the input data (default: sam).
• -r: for paired-end data, the alignment have to be sorted either by read name or by alignment position (default: name).
• -t: feature type (3rd column in GFF/GTF file) to be used, all features of other type are ignored (default: exon).
• -s: whether the data is from a strand-specific assay (default: yes).
• -m: 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 reads 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?