How to perform BLAST with selected genes from RNA-seq results to the reference sequence??

Question

[ongchip]

I have performed SNPeff with RNA-seq results. Finally, I want to design primers but I can't know the sequences near SNP positions. I want to compare the sequences of the selected transcripts within samples or SNP between samples and the reference genome. I don't know what files and programs should be used. fastq.gz files? SAM files? BAM files? or VCF files? Is there an easy way to perform this?

Answer 1

[Trivas]

You could use a combo of bedtools getfasta to get the sequence you're interested in then use samtools view to get the mapped reads that fall into that region. I would then convert the output of samtools view into fastq files then do a multiple sequence alignment over your reference sequence. This method assumes you know where your SNPs are (approximately).