I an doing RNA-seq on two closely related plant species (~98% similar in transcript region), one of which is sequenced.
My problem comes when I align the reads from the unsequenced species to the sequenced genome. Most of the reads align (at 2 bp mismatch level), however some are being rejected due to 3 or 4 bp mismatches. I would like to improve on the alignability of reads/reference genome to ensure the observed differences in expression between species are true (not due to differences in read alignability).
My idea is as follows:
Can anyone recommend programs or a strategy to do this, or even comment on the validity of the approach?
Any feedback/contribution would be much appreciated! Thank you!!
Note: the reads are very short: 40 bp.
My problem comes when I align the reads from the unsequenced species to the sequenced genome. Most of the reads align (at 2 bp mismatch level), however some are being rejected due to 3 or 4 bp mismatches. I would like to improve on the alignability of reads/reference genome to ensure the observed differences in expression between species are true (not due to differences in read alignability).
My idea is as follows:
- Align the reads to the reference (allow ~2 bp mismatch)
- Use the alignment to generate a consensus sequence, ie where there are SNPs between the reference and the reads, change the base on the chromosome to reflect that of the reads. Use stringent parameters so only true SNPs are 'corrected'.
- Realign the reads to the modified genome (allow ~2 bp mismatch). Now reads with 2 bp mismatches should align 'perfectly' and reads with up to 4 bp mismatches will align.
Can anyone recommend programs or a strategy to do this, or even comment on the validity of the approach?
Any feedback/contribution would be much appreciated! Thank you!!
Note: the reads are very short: 40 bp.
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