Can BWA align reads to references that are even shorter?

[asiangg]

We have obtained a lot of RNA-seq reads from HiSeq with 100bp in length. We are trying to align them to a splice junction database which only contain sequences that are 62bp in length. Is this possible? Are there any parameter values that need to be tweaked? Would soft clipping automatically mark both hanging 5' and 3' read fragments?

Hope some BWA guru can answer the above questions. Thanks!

[asiangg]

I'm trying to pop this post up for myself. Nobody had any experience with the above challenge?

[swbarnes2]

Sorry, never tried it. I used SOAP for bacterial alignments, and I know it seg faults when reference sequence is smaller than reads.

I recommend just trying it. If it doesn't work, maybe pad the 62 base junctions with N's.

[Sylphide]

I know I had some reads that were over-hanging from the end of the contigs (when mapping RNA seq onto CDS sequences) and it worked fine with BWA.

[swbarnes2]

On second thought, I think the way bwa works is it concatenates all the reference sequences into one long file. So if a read falls off the edge of one, and lands on another, that won't stop mapping.

However, what this means is that if your read starts a few bases upstream of reference junction B, and Junction A was concatenated to junction B such that the start of B is right after the end of A, samtools is going to report that your read is in junction A, becuase that's where it started. I think there will be a flag, or the MQ will be zero, something like that, so it would be catchable, but no easily, and it won't be easy to figure out. It's probably easier to pad your reference with n's so that this won't happen.