AMPure XP clumps during Cas9 library prep

Nereus · 09-28-2020, 12:49 AM

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?

gringer · 01-18-2022, 06:32 PM

Try needle-shearing your DNA prior to doing the CRISPR-Cas9 digest, e.g. 20 passes with a 26G or 29G needle (see here for more information). This has been working well for us to make the samples easier to handle and quantify.

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09-28-2020, 12:49 AM	#1
Nereus Junior Member Location: Zurich Join Date: Sep 2020 Posts: 2	AMPure XP clumps during Cas9 library prep 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?

01-18-2022, 06:32 PM	#2
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 843	Try needle-shearing your DNA prior to doing the CRISPR-Cas9 digest, e.g. 20 passes with a 26G or 29G needle (see here for more information). This has been working well for us to make the samples easier to handle and quantify.