I have a very specific use-case for short read alignment that I was hoping the community would be able to help me out with.
I am working on a project to detect errors in short (< 1Kb) synthesised DNA. We are using a variety of approaches to sequence these molecules including PacBio Hifi and MiSeq, and we are sequencing them very deep (~ 20k - 200k X). I have played around with bwa mem and minimap2, and while these both perform very well generally, they generate different results to those obtained by manual alignment of individual reads to the reference sequence (presumably this comes at the cost of blistering speed).
For example, in comparing the alignment of the same read (using the below parameters):
And then comparing with a manual alignment using NCBI BLAST:
bwa mem produces the result that I would expect.
For aligning PacBio HiFi reads I use the following parameters:
bwa mem -x pacbio <ref.fa> \ | samtools sort - \ | samtools view -q 30 -Sb - > s1_bwa.bam
minimap2 -ax asm20 --MD --cs --eqx <ref.fa> \ | samtools sort - \ | samtools view -q 30 -Sb - > s1_mm2.bam
Thus, I would like to know what the community would recommend in terms of most accurate read aligner, where speed is not at all a priority.
Is there a particular tool or algorithm that would be most appropriate?