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Old 11-29-2018, 04:27 AM   #1
lapensee
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Default Inefficient ATAC-seq library prep amplification

Hi all,

I’m having trouble generating an “optimal” library for sequencing.
I am using a human cell line derived from an adrenocortical tumor (from ATCC).

I have tried 12.5, 25, 50, and 100k cells with 2.5ul Tn5 (Illumina).
I've tried lysing with and without Digitonin, no difference.

qPCR determined that I needed ~5-7 extra cycles of amplification.

Qiagen cleanup was not removing the primer, and I had large peaks at ~2000 or bigger, so I switched to AMPure beads (double sided purification, first 0.5X then 1.8X)

I never seem to get those nice big peaks around ~<200, 400 just a blip. See pics below.

Does anyone have any ideas for improving the efficiency of my library amplification?

Thanks
Chris
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File Type: jpg ATAC-Seq library QC.jpg (92.5 KB, 86 views)
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Old 12-03-2018, 07:41 AM   #2
lapensee
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If not suggestions, would someone be willing to post a pic of a GREAT bioanalyzer profile (for ATAC-Seq)?
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Old 12-13-2018, 10:15 AM   #3
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Default Undertagmentation

Suspecting undertagmentation. Anyone else run into this problem, and if so, what was your remedy?
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File Type: jpg Undertagmentation.jpg (76.7 KB, 50 views)
File Type: jpg Undertagmentation2.jpg (89.9 KB, 56 views)

Last edited by lapensee; 12-13-2018 at 10:25 AM. Reason: Add figure
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Old 12-13-2018, 03:38 PM   #4
Meyana
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I have seen the same thing repeatedly with FACS sorted nuclei isolated from frozen tissue, hoping someone has an idea on how to optimize...
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Old 03-20-2019, 09:16 AM   #5
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FWIW I got this working. I adopted the OMNI-ATAC protocol and get great library amplification from 5k cells using only 2% the amount of Tn5 required for 50k cells. Adding a protease inhibitor during transposition was crucial for my library prep.
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Old 04-03-2019, 01:16 AM   #6
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Hey, did you use the new Nextera DNA flex Library Prep kit?
I've been having similar problems to you but nothing I do seems to work.
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Old 04-03-2019, 03:45 AM   #7
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I used "Nextera DNA Library Prep Kit"
FC-121-1030
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Old 08-10-2019, 08:15 AM   #8
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What protease inhibitor did you use in your protocol? I adopted the Omni-ATAC protocol but there's no qPCR amplification, even though I have DNA (7.6ng/uL obtained from 50,000 thymocytes).

I don't even know if my pre-amplification succeeded or not.
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Old 08-12-2019, 06:10 AM   #9
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Halt protease inhib without EDTA (100x) from thermofisher
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Old 08-12-2019, 08:53 AM   #10
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Well, after analyzing my qPCR results again, what I have to say is that the PCR looks awful but there's something there. Unfortunately, by the end of the 20 cycles I didn't hit a plateau stage and the amplification are of very low amounts. The exponential algorithm fails even.
Considering that I tried with the Omni-ATAC buffer with Tween and digitonin, I am considering re-trying only with NP-40 like in the original Buenrostro paper. Does anyone have any other idea of other optimization steps?
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Old 08-14-2019, 01:10 AM   #11
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By the way, I am doing the qPCR quantification with Power SYBR Green PCR Master Mix from Thermo Fisher instead of the NEB Next HF Master Mix.

Does anyone know if this might impact the qPCR quantification?
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Old 08-14-2019, 01:34 AM   #12
Meyana
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I started out using the PCR mix in the Nextera kit for both PCR and qPCR and got little to no amplification. Switching all steps to NEB (as described in the Omni paper) got everything working smoothly for me and highly reproducible with as low as 500 FACS'ed nuclei.
Based on this, I recommend using NEB products for all steps.
Also, I do not think it is a good idea to "switch" polymerase mix between quantification and amplification step.

Best of luck!

Last edited by Meyana; 08-14-2019 at 04:18 PM.
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Old 08-20-2019, 01:47 AM   #13
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Yup, I got everything working by using solely the NEBNext master mix.

Now, I'm just wondering about performing bead clean-up... I've decided to do a Fragmet analyzer before doing size selection, and depending on the profile I'll decide if I need to do a size selection or not.

By the way, does anyone have an example of a Fragment Analyzer profile of a successful ATAC-seq library?

Last edited by Rosmano; 08-20-2019 at 02:26 AM.
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Old 08-20-2019, 03:53 PM   #14
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I always do both upper and lower size selection using AMPure XP beads, I don't see a reason not to just do it right away (unless you are unfamiliar with bead cleanup and want to have a way to check you process).
I add 0.5x, keep supernatant, add 1.3x (total 1.8), keep beads, elute. Then check BA.

I attached some BA tracks from a test I did, D and E are how I like my libraries to look and these two sequenced nicely.
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File Type: pdf High Sensitivity DNA Assay_2019-02-13_004.pdf (1.74 MB, 4 views)
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