Hi guys,

I generated some primers by mutating amino acids in a DNA sequence of 1553 bases.. My primer structure is as follows;

19bp sequence(flanking sequence%100 match)+ 3 bp (mutated) + 19 bpsequence(flanking sequence %100 match)

Say i have some sequence that start with start codon. Following amino acids are mutated into other amino acids by changing codons. To show that I did this correct. I thought mapping to a reference (1553 bp DNA sequence) will show me those primers.

I followed these steps;

bwa index ref.fa
bwa aln tolc.fa primers.FASTA | bwa samse ref.fa - primers.FASTA | samtools view -bu - | samtools sort - > aln_sorted_output.bam && samtools index aln_sorted_output.bam

I am aware that I am swimming against stream by trying to decrease the specificity of that algorithm but couldnt think of a better way.

So my questions are;

1) Is my method practical ? if not how would you achieve this ? 2) (If my method is ok) What options in bwa should I tweak. I tried to lower the scores of gap penalty and mismatch penalties but did not work well.

Thank you very much for your help,

Best regards,

Tunc.