Question about authenticity of Addgene Human GeCKO Lentiviral sgRNA Library v2?

[Yuxiang Zheng]

Hi,
We obtained Human GeCKO Lentiviral sgRNA Library v2 from Addgene in March 2016. Electroporation, bacterial plating and maxi prep all went fine. But the Miseq result is very bizarre: most (>99%) of the sgRNA reads from Miseq could NOT be mapped to the original GeCKO library. Attached is a snapshot of the reads for GeCKO library A and B. Is this a different library? Anyone having a similar experience or suggestions?
Thanks,
Yuxiang

[Neville Sanjana]

Dear Yuxiang,

I wasn’t able to identify those sequences in the library and I too am puzzled by what they might be. Perhaps it is adaptor or PCR primer contamination? I haven’t heard of this problem before and there are many users of the GeCKOv2 library.

We have a recent Nature Protocols paper with detailed protocols for library sequencing that might be helpful: http://www.nature.com/nprot/journal/v12/n4/full/nprot.2017.016.html

In particular, did you see the correct (predicted size) bands after the PCR from the plasmid pool? And were the samples gel-extracted before sequencing?

Best wishes,

Neville

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[Indumati Sharma]

Dear Yuxiang,

I am curious to know what happened with your library. Were you able trouble shoot?

Thanks,

Indu

[Yuxiang Zheng]

Hi Indu,
We're currently troubleshooting. Our preliminary result shows that, after correcting a bug in the code (to take into account that an extra G is added to the 5' end of a subset of sgRNA sequences), now we can assign ~ 55% of the reads to the original GeCKO library. The coverage seems decent, with no underrepresented or overrepresented sgRNAs.
Yet we don't understand why ~ 45% of the reads cannot be assigned to the GeCKO library. Curiously, the top two, strongly enriched sgRNA hits in our positive screen actually map to non-coding regions of the human genome and are followed by NGG (PAM). Are these unintended sgRNAs the result of solid phase synthesis errors?
Yuxiang

[Indumati Sharma]

Dear Yauxiang,

Have you processed the reads before alignment? like removing 5' and 3'ends, N reads, reads that aren't exactly 20bp.....etc. Trying doing blast search for some of the reads (just randomly) to see what percentage of them map to the genome. Or probably try looking into the reads just in case you might be able to identify something unusual.

Can understand the frustration about reads not aligning. Once you do solve this problem please do post it in this forum. Would be really helpful for the rest.

Cheers,

Indu

[Yuxiang Zheng]

Hi Indu,
Thanks for the excellent suggestions! To elaborate on the previous post, we made a mistake while trimming the sequences: the extra G was left untrimmed. Yes, I'll post further progress here.
Yuxiang