What does this Warning in HTSeq-count means? (Warning: Mate pairing was ambiguous)

Question

[tiago211287]

After running HTSeq-count, I've got this warning. What does it means?

Warning: Mate pairing was ambiguous for 3121 records; mate key for first such record: 'FCC4UE9ACXX:6:2105:5458:19665#', 'second', '1', 12979515, '1', 12979588, 162).

[Ashutosh Pandey]

I think the other mate for this read is missing and as a result HTSeq is throwing a warning. Normally, if only one of the read from the pair gets mapped, the other unmapped read should be kept in the bam file and marked as unmapped. These unmapped reads may be missing from your bam files. May be you performed some pre-filtering on the bam file. Try searching for FCC4UE9ACXX:6:2105:5458:19665# read id in your bam file and if you only get one read then whatever I have explained above is right.

[tiago211287]

Well, Check this out:

Warning: Mate pairing was ambiguous for 5085 records; mate key for first such record: ('FCC4UE9ACXX:6:1314:2611:46102#', 'second', '1', 6230599, '1', 6230666, 155).

$ samtools view mix-18Aligned.out_sorted_merged.bam | grep -c "FCC4UE9ACXX:6:1314:2611:46102#"                   
4

$ samtools view mix-18Aligned.out_sorted_merged.bam | grep -c "FCC4UE9ACXX:6:2104:9464:13899#"
2

The read from warning got 4 entries instead of 2.

[lolec]

Hi! Did you manage to figure out why some reads have the same name? I think I have the same issue with my data.

[tirza.doniger]

You probably have some reads with secondary alignments.