what's wrong with htseq-count codes???

Question

[biologo]

hello, friends:

i was using the htseq-count to calculate the reads number, after run the tophat, then i isolate the uniqmapped reads, actually ,it was the paired-end reads, but i find uniqmapped reads only contain the single-end reads(no identical readsID),that is one thing that i fell confused??? next, i do like this:

samtools view -@ 5 -S uniqmap.sam -b -o uniqmap.bam
samtools sort -@ 5 -n uniqmap.bam -o sort.bam
samtools view -@ 5 -h sort.bam -o sort.sam
htseq-count -m union -s no sort.sam $GTF > union.out

and the result is nothing, err report that:

Warning: Read HWI-ST1434:279:HTFV7BCXX:2:1115:7046:27240 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read HWI-ST1434:279:HTFV7BCXX:1:1115:5605:41821 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read HWI-ST1434:279:HTFV7BCXX:1:2111:7809:19690 claims to have an aligned mate which could

can you help me figure it out???

Answer 1

[WouterDeCoster]

There is no need to filter the uniquely mapped reads yourself (likely the step which went wrong). Htseq count will be default only use read with mapQ >= 10.