Library preparation and NGS troubles

I am perfroming CRISPR-Cas9 KO screens with a custom library we cloned ourselves that contains pairs of guide RNAs (pgRNAs).

There are some problems I have been facing during the NGS steps, so I would like to ask you for a suggestion.. perhaps you might help, if possible.

We employ home-made primers that contain custom sequence for my plasmid, staggered nt, adaptamers for Illumina sequencing, so I can perform only one PCR step (25 cycles) to prepare the library.

We afterwards purify the product with AmpureBeads and send it for a sequencing trial even if we had some other products apart from the main one. we performed PE (Paired End) 150bp sequencing. the sequencing was ok, but given we were not completely satisfied with the % of mapped read, we decided to perform Gel extraction (in the same way and with the same kit you describe in the Nature protocol)

The problem we had here, was a drop in the quality of the Rv read, just at the end of the read (unfortunately where our guide is placed). In a previous trial withot gel extraction we haven't faced such a quality drop..

Do you think that the gel extraction can cause a drop in the quality of the read? is there any suggestion you hav in mind?



Thanks a lot in advance!

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Fuchsia Kelpieover 3 years ago

We have been gel extracting our PCR products prior to NGS without any issue with read quality. Is it possible that in your gel extraction you potentially left some contaminants that may have reduced the purity of the final product?

We also only sequence from 1 end for the NGS analysis to save reagents. Depending on your sgRNA target sequence position, you can sequence using Read 1 or Read 2 - I don't think it is necessary to do both.

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