Are you sure this is actually what you want to do? If you align reads from the whole genome to only chromosome 21, you will get false alignments. There are many regions with high similarity in the genome. For the purposes of illustration, let's imagine small regions on chromosome 1 and chromosome 21 that differ only by two base pairs. If you align only to chromosome 21, reads drawn from chromosome 1 will get matched up with their best alignment, which will be the highly similar region on chromosome 21. If you aligned against the whole genome, though, they would find a better (and correct) match on chr1. Thus, in many cases, it makes sense to align to the whole genome first, then subset out your chromosome 21 reads from the alignment file. If aligning to chr21 is really what you want to do, you should follow the instructions given by Sangwoo Kim - just create a new BWA index using only the chromosome 21 fasta. - Chris Miller