Hi, I am analyzing sncRNA reads (single-end) provided by
Nextflex sequencing method. I used
-m 1 option to align the reads to the mouse genome. Then I used
HTSeq-count to count the number of reads assign to each feature. I get low counts in
HTSeq-count and I do not know this is because of the alignment or the HTSeq-count (I did not specify
-s option so the default was yes).
And when I run
samtools flagstat file.bam on one of the bam samples I get these output.
733523 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 346170 + 0 mapped (47.19% : N/A) 0 + 0 paired in sequencing 0 + 0 read1 0 + 0 read2 0 + 0 properly paired (N/A : N/A) 0 + 0 with itself and mate mapped 0 + 0 singletons (N/A : N/A) 0 + 0 with mate mapped to a different chr 0 + 0 with mate mapped to a different chr (mapQ>=5)
I am very new to these kinds of analyses so could someone please help me to understand if the alignment is correct or not and there if is any problem with
HTSeq-count to be solved e.g.
-s option? Many thanks