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Old 04-04-2017, 02:07 PM   #1
duke of iredell
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Location: Southern US

Join Date: Apr 2017
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Default ATAC-seq question involving customized primers and adapters

Hello everyone,

I do not have a ton of experience with sequencing technologies, so I was hoping someone here could point me in the correct direction.

I am working on ATAC-seq and using the protocol from the Greenleaf and Chang labs from Stanford that I believe is common. It involves preparing the cells and performing a tagmentation reaction using the illumina kit FC-121-1030. A PCR amplification of the resulting DNA fragments is then performed using customized nextera primers that I ordered from Sigma. The resulting DNA is then purified using beads.

My question is about the customized primers- Ad1_noMX, a primer used in each reaction that appears to have no barcode- and barcoded primers (Ad2.1_TAAGGCGA, Ad2.2_CGTACTAG, etc.)

I am unsure of what adapters I should use for sequencing. I know that adapter kits exist such as TruSeq Nextera DNA adapters which have sequences similar to those of the barcoded primers I used, however this kit appears to use two different barcoded adapters i7 (the ones which would be appropriate for me) as well as i5 (ones that do not appear appropriate for me, since I used the Ad1_noMX primer).

From what I have read, there is an index kit which is sold by nextera that involves this second set of barcodes. I'm guessing that groups have developed this more customized protocol involving customized primers to avoid paying more money, but this is total speculation on my part.

Basically, I am unsure of which adapters to use for sequencing (specifically for the Ad1_noMX primer) and do not want to tell the people sequencing this something incorrect.

Does anyone have any experience with this out there who could help?
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