As a part of plant genotyping research and CRISPR cas9 mutation screening methods, I need to sequence and align many reads in order to capture low-frequency varients. therefore, im using NGS (rather then Sanger) Im trying to align reads from a fastq file (pair-end) to a short amplicon/reference which is about 200 bp.
I tried using the Bowtie2 aligner. first, used the Build option pretty generically:
bowtie2-build ref.fa ref
then do the alignment:
bowtie2 -x ref -1 R1.fastq -2 R2.fastq -X 200 --fr -S output.sam, which executed with no issues, though not one read was aligned...
Can anyone please explain what's going on? or even better, how to perform this kind of analysis.
Thanks in advance, Itamar