Hi Friends,
while I run my RNA-Seq data (Tophat/HISAT2 aligned output & samtools name sorted file) in HTSeq2 for counting DEG, which display warning message like “Mate records missing for 5069 records; Paired end read :HWI-D00486:7:1116:11181:7055” and “Mate pairing was ambiguous for 61656 records;” after SAM alignment record pairs processed. Kindly any one provide a solution to solve this warnings.
Some of your paired reads wee not at all / not well matched with their mate (the other read in a pair), so that HTSeq2 can not be sure that the fragment between the two reads was correctly amplified. Given its options, I guess it will not count those reads / fragments. It'a normal if it represents a (very) low percentage of your reads, but I think it's the case giben the numbers you indicate. How many total reads / fragments are you analyzing? Good luck!
Thanks Fabrice, The Illumina data contain 46 million reads after filtering for each samples. The script I used is "$ htseq-count -s no -r pos -t gene -f bam file.bam Reference > output.count". The reference Gtf/gff3 file contain 27987 gene annotation information. HTSeq2 default overlap resolution mode 'Unioin' is used for analysis. Is there any parameter (functions) I want to change in my script for run
Easiest solution is to not use paired-end reads. This is not necessary for pure counts-based RNAseq (as opposed to differential splicing etc.)
- Badges
- Science method