Issue with Alignment Results Using Soft-Masked Genome in Bismark

[Chanspace]

I am currently conducting Whole Genome Bisulfite Sequencing (WGBS) data analysis using Bismark and plan to utilize a soft-masked genome, where all repetitive and low-complexity regions are marked with lowercase letters.

During the index generation step, I observed that the index created is consistent with the unmasked genome. However, I noticed a significant difference in the results during the alignment step, specifically in the number of uniquely aligned reads. It appears that tools like Bowtie2 ignore the soft-masking, treating the lowercase letters as uppercase during alignment.

Is there a specific parameter or approach in Bismark that would allow me to achieve alignment results with the soft-masked genome that are comparable to those obtained with the unmasked genome? Any guidance or advice would be greatly appreciated!

Thank you!

[FelixKrueger]

To be perfectly honest, I don't exactly know whether or not Bowtie2 treats soft-masked genomes differently to unmasked genomes but I don't think it does (Google also doesn't seem to know, "how does Bowtie2 treat soft-masked index" didn't yield any great insights either).

What would you like to achieve by soft-masking repeats?

[Chanspace]

I'm sorry, I may not have expressed myself clearly. What I actually want to know is how to ensure consistent detection rates when using unmasked and soft-masked genomes in Bismark. The reason is that we have utilized soft-masked genomes in other omics analyses, so we hope to maintain consistency. However, we compared unmasked and soft-masked genomes in WGBS data analysis with bismark, and even though the generated indexes are the same, there are still differences in the subsequent methylation detection rates.

[FelixKrueger]

I am afraid I don't really have an answer you, I would have to do some tests with reproducible examples. I think your best bet would be to cross-post this questions over at the Bowtie2 repo - as the effects of this behaviour will likely be part of the Bowtie2 strategy with soft-masked indexes. I you get an answer, I'd be curious to learn more details. Sorry if this is not immediately useful.