As a preparation step before aligning my RNA-seq reads to my reference genome, I am trying to generate a mappability mask. I know that masking the genome before aligning RNA-seq reads is not conventional, but we are doing that to facilitate the alignment of the RNAseq reads to highly similar gene families, which are an important focus of my study.
I am using the code from SNPable (lh3lh3.users.sourceforge.net/snpable.shtml) which uses bwa as the default aligner. I can generate my mask genome perfectly using bwa, however, when I try to use a slice-aware aligner like STAR or HISAT2, I cannot generate the mask.
After having a look at my SAM files, I suspect that the problem comes from the lack of information regarding the suboptimal hits in my STAR SAM files (X1 flag in bwa). However, I cannot find the equivalent information using STAR, so I cannot get the SNPable code to work.
I am working with an eukaryotic organism and therefore I require to use a splice aware aligner for my RNA-seq reads, for this reason, I cannot use bwa as my aligner. Ideally, I would like to generate the mappability mask with the same aligner that I would use later on, for this reason, I am trying to find a way to generate the mappability mask with a splice-aware aligner.
Does anyone has any suggestion about know how I could:
1) Modify the STAR SAM files/code to make the mappability mask using STAR as my aligner?
2) Can suggest a splice-aware aligner that gives suboptimal hits information? Or an alternative program to generate the mappability mask?
Any help would be very appreciated 🙂