NGS 0 Match

I performed a HiSeq on my amplified lenti library and some transduced cells to determine representation and I'm getting 0 matches.

I checked for sequence correspondence with the published sgRNA library with no success, suggesting that either the amplification or NGS were incorrect and not just a misalignment.

Amplification looked to be correct with gel electrophoresis with a single band at 270~ bp but our genomics core told me that when the did the RT-qPCR they actually got a 400 band for the amplified library and 300 band for the transduced cells. I use the primers from the Joung et al Nature 2017 paper.

This is likely the area of concern but not fully sure if this is a contaminant or something else? I can find the "CGAAACACC" before the guide and the NGS worked making me believe that it the primers I used had the Illumina adapters and construct altogether.

So I'm stumped. Do you guys have any suggestions?

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Fuchsia Kelpiealmost 5 years ago

Since you can find the "CGAAACACC" sequence before the guide, can you check the target sequences against the sequences in the library, and figure out what target sequences you are getting? This can help you narrow down whether this is an analysis issue or a contamination. Keep in mind that the SAM human library does not have a 'G' inserted before the gRNA target sequence, but the GeCKO human library does.

It is also concerning that you are getting a 400 band for the amplified library, because this could indicate potential contamination for the library.

Lavender Gnomealmost 5 years ago

I foolishly thought I could get away a 1x50 but was a little short so its hard to make any definitive conclusions so I'll have to run it again. Can I ask, what type of target sequences would indicate a analysis issue vs contamination issue? There are a handful of target sequences that are sufficiently sequenced but do not correspond to any of the library sequences. It just seems odd that I'm able to get the rest of the sequences...

A separate question is do you notice a significant difference between the NEBNext polymerase vs the Phusion?

Fuchsia Kelpiealmost 5 years ago

If you can find the end of the U6 promoter sequence, but the target sequence does not match anything in your library and instead matches something you have worked with, then it is likely a contamination issue. If you can find the end of the U6 promoter sequence, and you can see target sequences that match sequences in your library, then it is likely an analysis issue. The parameters in the output of the library analysis script should help inform how to resolve any potential issues.

I haven't done a direct comparison between NEBNext and Phusion, but as long as it's a high fidelity enzyme, then it shouldn't be a problem.

Lavender Gnomeover 4 years ago

Thank you for all the help so far!

I have basically manually performed the analysis that the python script has done and am able to find the end of the U6 promoter (CGAAACACC) and the subsequent gRNA scaffold (gttttagagctagaaatagcaagtta...) with 20 bp (some 21 bp) sgRNA inbetween just as described.

We've never done any sgRNA work in my lab and when blasted nothing corresponds to any constructs we've made in the lab but rather random chromosomal snippets which I presume were targeted.

So now I'm just confused! The library I'm using is from the addgene site and when I manually search for correspondence, no dice.

Has anyone come across this issue?

Lavender Gnomeover 4 years ago

Figured out the issue. I did double read (as it was the quickest and cheapest at the time) and it turns out I needed to do the inverse complement.

Fuchsia Kelpieover 4 years ago

Glad to hear you figured it out - good luck!

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