htseq-count low count problem

[gandalf886]

Hi guys,

I am looking at some RNA-seq data using DEseq2, before this I need to get a count table for each gene.

here is what i have done:

mapped the stranded, paired-end reads to transcriptome using tophat2:

tophat -p 12 -r 60 -o $out --transcriptome-only --no-novel-juncs --no-coverage-search --library-type fr-firststrand --transcriptome-ind
ex=$known $hg19 lane1.1.repaa_val_1.fq lane1.2.repaa_val_2.fq

I got about 6.5 million mapped pairs, which is about 50% of the input reads.

then i took the mapped reads and count them against a gtf table

htseq-count -f bam -r pos -t exon -i gene_id accepted_hits.bam hg19.gtf > accepted_hits.bam.counts

I got 0.4 million reads counted into the table and the number of no feature reads is about 13 million.

I tried to sort the bam files using samtools and use -r name option in the htseq-count line but it also didn't work.

this is how the gtf file look like

chr1	unknown	exon	11874	12227	.	+	.	gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS14844";
chr1	unknown	exon	12613	12721	.	+	.	gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS14844";
chr1	unknown	exon	13221	14409	.	+	.	gene_id "DDX11L1"; gene_name "DDX11L1"; transcript_id "NR_046018"; tss_id "TSS14844";
chr1	unknown	exon	14362	14829	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	14970	15038	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	15796	15947	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	16607	16765	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	16858	17055	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	17233	17368	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	17606	17742	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	17915	18061	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	18268	18366	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	24738	24891	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	29321	29370	.	-	.	gene_id "WASH7P"; gene_name "WASH7P"; transcript_id "NR_024540"; tss_id "TSS7514";
chr1	unknown	exon	34611	35174	.	-	.	gene_id "FAM138A"; gene_name "FAM138A"; transcript_id "NR_026818_1"; tss_id "TSS8403";
chr1	unknown	exon	34611	35174	.	-	.	gene_id "FAM138F"; gene_name "FAM138F"; transcript_id "NR_026820_1"; tss_id "TSS8403";
chr1	unknown	exon	35277	35481	.	-	.	gene_id "FAM138A"; gene_name "FAM138A"; transcript_id "NR_026818_1"; tss_id "TSS8403";
chr1	unknown	exon	35277	35481	.	-	.	gene_id "FAM138F"; gene_name "FAM138F"; transcript_id "NR_026820_1"; tss_id "TSS8403";
chr1	unknown	exon	35721	36081	.	-	.	gene_id "FAM138A"; gene_name "FAM138A"; transcript_id "NR_026818_1"; tss_id "TSS8403";
chr1	unknown	exon	35721	36081	.	-	.	gene_id "FAM138F"; gene_name "FAM138F"; transcript_id "NR_026820_1"; tss_id "TSS8403";
chr1	unknown	CDS	69091	70005	.	+	0	gene_id "OR4F5"; gene_name "OR4F5"; p_id "P1230"; transcript_id "NM_001005484"; tss_id "TSS14428";
chr1	unknown	exon	69091	70008	.	+	.	gene_id "OR4F5"; gene_name "OR4F5"; p_id "P1230"; transcript_id "NM_001005484"; tss_id "TSS14428";
chr1	unknown	start_codon	69091	69093	.	+	.	gene_id "OR4F5"; gene_name "OR4F5"; p_id "P1230"; transcript_id "NM_001005484"; tss_id "TSS14428";
chr1	unknown	stop_codon	70006	70008	.	+	.	gene_id "OR4F5"; gene_name "OR4F5"; p_id "P1230"; transcript_id "NM_001005484"; tss_id "TSS14428";

Any ideas? thanks

[dpryan]

Have a look at things in IGV or use the -o option to track what's happening to reads that aren't getting counted but you think should be.

[gandalf886]

Got most mapped reads counted if I specify -s no, but the library was made using a strand-specific protocol and mapped using tophat in --library-type fr-firststrand mode. Don't know why.

[dpryan]

I'd have to recheck the strandedness settings, maybe you just need "-s reverse" to match your library type.