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Old 12-14-2015, 02:48 AM   #1
Location: UK

Join Date: Nov 2015
Posts: 19
Default HTseq - count - high ambiguous count rate and reads with missing mate encountered

Hi. I'm using HTseq to produce some count read files that I can then use for NOIseq and edgeR. I used trimmomatic to trim my reads in paired end mode and merged the paired ends in tophat2 using

/home/user/Downloads/tophat2/tophat -p 4 -o /home/user/Desktop/sam/trimmomatic_out/2B82 --mate-inner-dist 100 --library-type fr-firststrand /home/user/Downloads/bowtie2/example/index/DinoAnt /home/user/Desktop/sam/trimmomatic_out/2B82/output_forward_paired.fastq.gz /home/user/Desktop/sam/trimmomatic_out/2B82/output_reverse_paired.fastq.gz
I then sorted them using the samtools sort function. When I run the resulting bam files through HTseq count function, I get a really high percentage (95%) of reads with missing mate pair and also around 65% amiguous feature rate.

I used this command and ran it on a gtf file I produced myself in cuffmerge from the RNAseq data I collected.

htseq-count -m union -s no -t exon -f bam -i gene_id /home/user/Desktop/sam/trimmomatic_out/10G87/accepted_hits.sorted.bam /home/user/Desktop/new_dino_merged.gtf
Can anyone help me why I am getting such high ambiguous count rate and such a high percentage of missing mate pairs?


Last edited by 4galaxy7; 12-14-2015 at 04:19 AM.
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