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I'm using HISAT2 (version 2.0.5) to try and find novel splice sites. Going through the SAM file it produces suggests that there are a number of alignments supporting the existence of splice junctions that are not being reported (i.e. the aren't in the file you get from using the "--novel-splicesite-outfile" option).
For example, the SAM record for one of the reads is:
SRR360120.14138165 83 V 12814114 60 9M1297N91M = 12813953 -261 ATCCCATGTCTTAATTAAACTTGTGGTAACTTTTAATGAATTAAACTTCTGATTTTGCCGATAAGCATATCATATGAAAAATACTAAAAATGTCGAAATGCC?CCCCDECEEEEC?D@FFDEECHHGEEGDEFHAGGIGJIIIHHGIJIGHCGDIJJGIIGHHCG@E?A9EAGIEGF@HHBFAJIGJHFF<FADBFF@@@ AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YS:i:251 YT:Z:CP XS:A:- NH:i:1
The way it's split suggests that there is a splice junction at V:12814123-12815419, but there's no such entry in the file of reported splice sites.
For comparison, I ran TopHat (version 2.1.0) and it produced the same alignment for this read. The difference is that TopHat DOES report the expected splice junction at V:12,814,123-12,815,419.
So, is there some criteria I'm missing that causes HISAT2 to not report certain splice junctions, even if there are alignments supporting them?
For example, the SAM record for one of the reads is:
SRR360120.14138165 83 V 12814114 60 9M1297N91M = 12813953 -261 ATCCCATGTCTTAATTAAACTTGTGGTAACTTTTAATGAATTAAACTTCTGATTTTGCCGATAAGCATATCATATGAAAAATACTAAAAATGTCGAAATGCC?CCCCDECEEEEC?D@FFDEECHHGEEGDEFHAGGIGJIIIHHGIJIGHCGDIJJGIIGHHCG@E?A9EAGIEGF@HHBFAJIGJHFF<FADBFF@@@ AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YS:i:251 YT:Z:CP XS:A:- NH:i:1
The way it's split suggests that there is a splice junction at V:12814123-12815419, but there's no such entry in the file of reported splice sites.
For comparison, I ran TopHat (version 2.1.0) and it produced the same alignment for this read. The difference is that TopHat DOES report the expected splice junction at V:12,814,123-12,815,419.
So, is there some criteria I'm missing that causes HISAT2 to not report certain splice junctions, even if there are alignments supporting them?