Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation

Similar Threads
Thread Thread Starter Forum Replies Last Post
Rn/cycles: qPCR decided ATAC-seq library amplification zhaolin Sample Prep / Library Generation 9 08-14-2019 01:48 AM
qPCR for determining ATAC-seq amplification cycles? anish_dattani_zoo Sample Prep / Library Generation 0 01-17-2018 11:19 AM
Atac-Seq - Side qPCR question - cycle number JP_seq Sample Prep / Library Generation 0 11-16-2017 06:34 AM
Why sub-saturation qPCR in ATAC-seq but not other seq methods? kropak1807 Sample Prep / Library Generation 2 06-21-2017 07:26 AM

Thread Tools
Old 01-27-2020, 02:46 PM   #1
Junior Member
Location: New York

Join Date: Jan 2020
Posts: 1
Default ATAC-seq, qPCR is not working.

Hi all,

I have been successfully doing ATACseq until I started doing PCR amplification by myself. So far, I just gave tagmented DNA to core for PCR amplification and QC. These samples showed good distribution of DNA fragment size.
However, I decided to do everything by myself to reduce to cost.

The problem is the qPCR for determining additional cycles is not working.
Here are what I did.

After transposase reaction, DNA yields were 0.5~2ng/ul in total 20ul.
I ran 5 cycles of pre amplification as described in omni ATAC seq.
All primers were from Sigma in normal quality.

First I tried sybr mix and i5 i7 primer mix included in Kapa quantification kit, but didn't work.
Even with the primer I used for pre amplification, it didn't work.
There was amplification on standard DNA, so I don't think the reagent is defective(also it's new).

So I sticked to original omni-ATAC protocol which uses 0.65uM of primers, the same polymerase I used for the pre-amplification, and add Sybr green. Still, it didn't work.
I tried with 10x primer mix from the KAPA, but it didn't help.

So I just ran additional 10 cycles more for every samples, submitted them to the core for QC.
Surprisingly, half of samples were showing good enrichment of sizes around 150bp - 400bp, and the other half was only having large fragments ( > 2000 ).

it seems like although I have good tagmented DNA, somehow my primers are not constantly binding to adapter sequence of fragmented DNAs.
Has anyone had quality issue with primers?

Still, it doesn't explain why those good samples are not amplified in qPCR with both i5 i7 primer mix and the custom primers I used for pre amplification.

Could running qPCR on 384 wells be problem?

what are those huge size of DNA fragments after amplification seen from bioanalyzer data?

I would appreciate any comment from you guys. Thanks !!
jinkyustar is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 08:47 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO