Hi all,

After stumbling on this post: https://bioinformatics.stackexchange.com/questions/16112/precisely-clipping-bam-file-to-bed-coordinates, I found out about the samtools ampliconclip package and wanted to use it for my own project. However, I'm running into some issues with using the samtools ampliconclip --soft-clip versus --hard-clip option.

Essentially, what I'm trying to do is clip certain reads of interest in a bam file down to a specific window that I specify using a bed file. However, when I run ampliconclip with --soft-clip, it gives me what I want, but when I run it with --hard-clip, it basically produces a blank bam file as output.

This is a picture of the unprocessed bam file of interest

This is the complement bed file that I want to clip

9   0   33261120
9   33262737    149000000000

After running the code samtools ampliconclip --both-ends --soft-clip -b input.bed input.bam> output.bam I get the result I want, which is all my reads clipped besides a certain genomic interval

However, what I really want is the hard clipped version of these reads. However, when running the same code samtools ampliconclip --both-ends --hard-clip -b input.bed input.bam> output.bam I basically get an empty file with no reads at all.

From my understanding, using ampliconclip with --hard-clip versus --soft-clip should only change the CIGAR string and not how the intersection with the bed file should work, so I'm a little stumped as to why its giving me such different outputs. Any help is greatly appreciated!