Hi everyone,
I'm using the ONT CRISPR-Cas9 targeted enrichment protocol (https://www.nature.com/articles/s41587-020-0407-5) to sequence a 18kb locus in DNA which I extracted from human brain using the Circulomics Nanobind kit (which is recommended for this application). The protocol has worked well on a few occasions (around 120x coverage, which I understand is all right for this application), but on other occasions the coverage was quite poor (e.g. 6x), despite the DNA being very high quality (>60kb on Genomic Tapestation).
I suspect that the low coverage stems from a problem during the library prep:
After Cas9-directed cleavage of DNA and ligation of ONT adapters, the library is purified with 0.3x AMPure XP beads. I have observed on several occasions that the beads form insoluble clumps the moment that they are added to the tube, and I suspect that much of the library is lost within the clump. I have tried prolonged incubation and gentle agitation, but I couldn't dissolve the clump, neither in the ONT Long Frament Buffer, nor in the elution buffer.
Has someone got any tips on how to avoid this issue?
I'm using the ONT CRISPR-Cas9 targeted enrichment protocol (https://www.nature.com/articles/s41587-020-0407-5) to sequence a 18kb locus in DNA which I extracted from human brain using the Circulomics Nanobind kit (which is recommended for this application). The protocol has worked well on a few occasions (around 120x coverage, which I understand is all right for this application), but on other occasions the coverage was quite poor (e.g. 6x), despite the DNA being very high quality (>60kb on Genomic Tapestation).
I suspect that the low coverage stems from a problem during the library prep:
After Cas9-directed cleavage of DNA and ligation of ONT adapters, the library is purified with 0.3x AMPure XP beads. I have observed on several occasions that the beads form insoluble clumps the moment that they are added to the tube, and I suspect that much of the library is lost within the clump. I have tried prolonged incubation and gentle agitation, but I couldn't dissolve the clump, neither in the ONT Long Frament Buffer, nor in the elution buffer.
Has someone got any tips on how to avoid this issue?
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