i am interested in the unmapped reads from it.I am looking for a very strict parameter like having 100% alignment match of reads to genome for it to be considered valid mapping. could anyone please suggest which parameters to be used for the same.
Looking forward,
Thnx
In my opinion, in this case, the selection of alignments should be done by filtering existing alignments and not by "forcing" the aligner to behave a certain way. Look into the SAM format specification and note how imperfect alignments are annotated.
For example, the NM tag will contain the value of the so-called edit distance to the reference. When that value is zero the query matches perfectly.
If, as in your definition, every alignment with a change should be called unaligned then filter for the NM tag value that is larger than 0 and select your aligments that way. If retaining a BAM file is essential you can even flip the flag to be unmapped.
Check out the penalty parameters --mp, --rdg and --rfg which you can set to something like 10000, the number of allowed seed mismatches -N which you can set to 0 and the option --very-sensitive to enforce stringent end-to-end alignment (the latter actually already contains seed mismatch of 0). Together these should discard every non-perfect alignment. Additionally you could be stringent with the post-filtering towards MAPQ to only keep very unique reads, e.g. with samtools view -q 30.
It may also help to add some context for why you want to do this? There may be a better option instead of doing this.