Advice for Stagger Diversity in NGS-Lib Fwd primers (Nature Protocols 2017paper)

Thank you very much for your recent protocol paper for CRISPR screening. I have couple of questions about the NGS primers for amplifying the sgRNA library.

1) We are planning to deep-sequence both mouse and human GECKO libraries (2-vector system) to check sgRNA representations in the same Miseq run via multiplexing each library with different NGS-reverse primers. I was just wondering while performing NGS, can I just mix each of 10 NGS-Fw (stagger) and use different NGS-Rev (barcoded) for each library (use mix of NGS-Fw(10 different primers) and change NGS-Rv for Mouse library A (Rv1) and B(Rv2), and Human library A (Rv3) and B (Rv4))?

2) After reaction is complete, can I pool each of 4 different PCR product and purify and gel extract and then quantify with Quibit? We are also planning to use 10% Phix control in this case.

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Fuchsia Kelpieover 5 years ago

I'm happy to hear that you found our recent Nat Protocols paper for CRISPR screening to be helpful. To answer your questions:

1. When amplifying sgRNA libraries for NGS, I usually run the 10 NGS-Fw primers as 10 separate reactions with a NGS-Rev barcode for each library. The PCRs in theory should also work if you mix the 10 NGS-Fw primers, but I tend to separate them because it is cleaner and reduces potential bias.

2. I usually pool all PCR products in the same condition (i.e., same barcode), purify, and quantify. Then if there are any significant differences in product yield between each condition, I will normalize and then gel extract to make sure that each condition will have similar numbers of NGS reads.

Hope this helps!

Violet Incubusabout 5 years ago

Im a little bit lost with the PCRs for NGS. In the paper from Nature (2017), in the Table 3 you provide 10 Fw primers and 8 Rev primers. Does it mean that we need to perform 10 PCR with each Fw and 1 Rv and then another 10 PCR with 10 Fw and another Rv until we complete the 8 Rev primers?

Fuchsia Kelpieabout 5 years ago

For the NGS primers, the number of reverse primers you will need to run depends on the number of screening conditions in your library since that determines the number of barcodes that you will need. For example, if you have 2 screening bioreps, each with an experimental and control condition, then you have 4 total screening conditions and you will need to run your PCRs for each screening condition with a different reverse primer so you can sequence on the same machine (NextSeq or HiSeq). I would recommend using all 10 forward primers for each condition to diversify the NGS library.

When you are doing QC on the sgRNA library, you will only need to use 1 reverse primer because you only have 1 condition.

Ruby Ghostabout 5 years ago

Do you run separate PCRs for each of the Forward Primers or would you mix the Forward primers to make it easier, especially if you running 96 PCR reactions per sample.

Fuchsia Kelpieabout 5 years ago

Both methods work. I currently have my forward primers stamped out in a plate so it is easy to multichannel them into the PCR individually.

Navy Trollover 3 years ago

this is a very interesting and apparently knowledgeble forum, so hopefully someone might be able to help me with my question:

I performed a CRISPR screen and eventually filled an entire lane of a flowcell for NGS. The sequencing run was very bad unfortuanelty, due to the very high similarity of my sequences, i.e. resulting from always sequencing a constant part in front of my sgRNAs first. However, I have used staggers only in a fraction of my samples, while the other samples contained 5 random nucleotides at the beginning of the sequenced DNA stretch. I am now wondering if having staggers in all samples would solve this problem and if anyone could tell me if you did ever run an entire lane filled with sgRNA library samples amplified with staggers?!

Thank you very much for any help.

Fuchsia Kelpieover 3 years ago

Using forward primers that stagger your sgRNAs will resolve your problem (see Joung Nat Protocol 2017 for the primer sequences).

Alternatively, if you are not able to re-prep your library, you can spike in 30-50% PhiX to add diversity to your library.

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