PCR Bias and Depth of Coverage for NHEJ Quantification with NGS

23 May 2017 1 8K Report

Hi,

I would like to use NGS to quantify the levels of NHEJ after Cas9 cutting of a genomic locus. I have gDNA from pools of transfected cells, on which I run a PCR#1 of about 150bp for sequencing (including overhangs for barcode addition (PCR#2)). I have 2 questions regarding this:

PCR cycle bias: in a pilot experiment of only 2 samples, I've noticed that the number of amplification cycles in the genomic PCR#1 affects the levels of NHEJ quantified from the NGS data. The more cycles, the less editing I observe, which suggests a positive selection for the WT allele. This makes sense, and is known from NGS for library validation, but I could not find any guidelines or recommendations for optimising this. Do any exist? I plan on doing a PCR cycle "titration" first, to see if it plots on a curve and if an optimal amount can be deduced. Obviously I will use the same amount of cycles for all experiments consistently, but do you have any suggestions for this?

Depth of coverage: how should depth of coverage be calculated for proper representation of level of editing? Most resources seem to focus on WGS/WES, RNA-seq, etc., as opposed to targeted seq. of a pool of cells.

I'm using the Illumina HiSeq2500 on HiSeq Rapid Run mode, if it's relevant.

Thanks,

Yuval

Badges
Science method

More Yuval Novik's questions See All

How can i predict the phenotypic effect (survival curve) of specific mutation/VUS?

we identified multiple mutations (vus) in human genes that we believe have a protecting effect against cancer. is there any database or web tool that can be used to detect the function (and...

23 November 2016 4,944 3 View

Why should we choose deep learning to machine learning? Why is CNN better for image classification? Can I get Typical Answer?

What Characteristics makes CNN work better?

03 March 2021 1,458 4 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

Anyone plz suggest relevant topic for research in image processing+Deep learning.?

I have selected brain tumor images ...but now found that already lots of research done n this topic.

03 March 2021 5,774 3 View

How to increase a model accuracy ?

dear community, my model is based feature extraction from non stationary signals using discrete Wavelet Transform and then using statistical features then machine learning classifiers in order to...

03 March 2021 6,994 5 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

What is the difference between L1 and L2 regularization in machine learning?

L1 and L2 regularization

28 February 2021 4,187 3 View

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View

Has anyone used the Illumina ITS1 primer sets?

Hi everyone, Illumina provides a list of primers to amplify with high taxonomic coverage the ITS1 region for further fungal sequencing, but I cannot find the exact amount necessary of each...

25 February 2021 6,969 3 View

How do you mesh a thin solid in Fluent?

Hello, I have used the shell function on SpaceClaim to create thin solids for the model I am studying. These are contained within an enclosure for my study. I chose this approach as it avoids the...

24 February 2021 8,856 3 View

Errors in DNA sequences?

I am trying to better understand the scope of DNA replication and sequencing errors, e.g. 1. I have seen similar error rates of 10e4 to 10e5 for cell & instrumental DNA replication,...

24 February 2021 4,397 3 View