I was following DESeq2 manual to process my simple RNASeq paired end data that involves wil type and stress treated plant.
I ran feature count using HTSeq (version 0.9.1) with the command,
htseq-count -a 10 -s 'no' WT-CON.sam /home/exp/DESEQ2/genes.gtf > WT-DESeq.txt
I noticed a Warning: 53525476 reads with missing mate encountered.
100000 GFF lines processed. . 604523 GFF lines processed. Warning: Read K00171:29:H2NYHBBXX:8:1128:23045:10019 claims to have an aligned mate which could not be found in an adjacent line. 100000 SAM alignment record pairs processed. . . 56400000 SAM alignment record pairs processed. 56500000 SAM alignment record pairs processed. Warning: 53525476 reads with missing mate encountered. 56509150 SAM alignment pairs processed.
samtools sort -o WT-CON.bam /home/exp/DESEQ2/WT/accepted_hits.bam samtools view WT-CON.bam > WT-CON.sam
Am not sure how can I overcome the warning. Do I need to sort BAM with -n and run HTSeq again or any other parameter is missing?