biased mutation calling/false negatives from HALOPLEX datasets?

[mmwmm]

Hi everyone,

I'm wondering if someone has an opinion/experience with that. Using Haloplex, considering the frequent occurence of restriction events in order to generate relatively small "sequencable" fragments from a whole genome, don't you run a high risk to actually CUT your mutated position (if they introduce a novel recognition site for the restriction enzymes)?
Wouldn't this completely obscure the aim for discovery mutational screening without prior knowledge of potential mutations?
Further, if the A but not the B allele of a heterozygous SNP is recognized by one of the restriction enzymes, wouldn't that also bias the allele-frequency of adjacent mutations on the same amplicon?

Best,
Max

[loretta.manning]

HaloPlex info

A key feature of the HaloPlex target enrichment system is that there are multiple unique amplicons (up to 8) designed to cover each base and the input gDNA is digested in 8 separate digestions using different enzymes. This is very helpful in preventing complete amplicon dropout in a situation where a mutation introduces a novel restriction enzyme recognition site due to the presence of other amplicons that can still capture the target region. In the same way, amplicon redundancy also mitigates allele bias.

Please feel free to contact us at [email protected] and we will be happy to answer your questions about HaloPlex.

[mmwmm]

Thanks for the extensive explanation. I would also be interested in real-world user experiences - maybe someone compared their results with other library-prep approaches? Any input would be very welcome!

[Zaag]

My problem with HaloPlex is that for quite a few of our targets they are unable to design multiple amplicons so you end up with the problems you describe in the first post. But in theory and in salespitches it seems like a really nice technology.