I'm trying to BWA to align my custom-designed sequences to a reference genome I generated corresponding to all the sequences present in the same panel. This is following the lentiMPRA protocol linked here https://www.nature.com/articles/s41596-020-0333-5#Sec1
The difficulty is that half of my sequences are exact reverse complement to the other half, which is considered in the pipeline published. If I simply do the default alignment, the pairs of sequences will not be easy to separate other than fishing the out using sam flags, which may not be compatible with downstream pipeline.
I'm trying to think if there's a way that I can create my reference genome with reverse complement sequences present but with a separate label. However, I couldn't figure out how to force BWA to align only the R1 reads to the forward strand of the genome, and R2 to the reverse strand. It sounds doable, but I couldn't find the argument in BWA. I suppose bowtie2 can work as well, but I'll have to make some other changes to the published pipeline.
Thanks! Le
Simply adding the reverse complement into the reference genome then filtering out the bam file with the wrong orientation will screw up MAPQ, which will be 0.