Bioanalyzing long ATAC-Seq libraries

JoeKutch · 08-08-2018, 01:09 PM

Hi All,

I am part of a sequencing core, trying to QC a client's ATAC-Seq libraries via the bioanalyzer on a DNA high sensitivity chip. Prior to running the samples I checked the concentrations with a qubit fluorometer and diluted them all to ~4-6 ng/uL. Perhaps a little high for the high sensitivity chip, but not enough to overload it.

After talking with the client I believed the problems was long fragments carrying over between samples, so I tried to space them out, only loading samples in position 1, 4, 7, and 10. Unfortunately, I am still getting garbage traces after the first sample. Does anyone have any recommendations for how to handle these samples?

I've attached the traces as an example - you can see the characteristic ATAC peaks in sample 1, and then some higher molecular weight material near the marker, continuing into the next two traces.

pmiguel · 08-10-2018, 09:18 AM

We had some Nextera libraries that looked like this recently -- turned out they were 10X overloaded.

Also, they are a client's ATAC-seq library? We recently had a set of these where the client had done and ampure upper cut and given us the supernatant without further clean-up! So they were somewhere north of 1M NaCl plus whatever PEG was in the ampure. We pulled the library down by adding more ampure, followed by the normal ampure washing/eluting the beads-- then everything looked fine.

--
Phillip

JoeKutch · 08-10-2018, 09:31 AM

Thanks for the reply Philip:

I don't think they are overloaded, but I can dilute further and try them again with some other samples. Qubit and then diluting should have prevented that.

They are a client's libraries - the ATAC protocol he used does not call for any clean-up, so there aren't any PEG/salts in them. After finding that out, we recommended a bead uppercut with 0.4x beads followed by 2.5x to recollect the libraries. He gave us a very limited amount for QC or I'd try it myself. Once he brings those newly cleaned libraries I'll update.

Thanks again for the help!

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08-10-2018, 09:18 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	We had some Nextera libraries that looked like this recently -- turned out they were 10X overloaded. Also, they are a client's ATAC-seq library? We recently had a set of these where the client had done and ampure upper cut and given us the supernatant without further clean-up! So they were somewhere north of 1M NaCl plus whatever PEG was in the ampure. We pulled the library down by adding more ampure, followed by the normal ampure washing/eluting the beads-- then everything looked fine. -- Phillip

08-10-2018, 09:31 AM	#3
JoeKutch Member Location: Philadelphia Join Date: Oct 2016 Posts: 17	Thanks for the reply Philip: I don't think they are overloaded, but I can dilute further and try them again with some other samples. Qubit and then diluting should have prevented that. They are a client's libraries - the ATAC protocol he used does not call for any clean-up, so there aren't any PEG/salts in them. After finding that out, we recommended a bead uppercut with 0.4x beads followed by 2.5x to recollect the libraries. He gave us a very limited amount for QC or I'd try it myself. Once he brings those newly cleaned libraries I'll update. Thanks again for the help!