Can't sequence ATAC-Seq libraries

favwiz · 10-03-2017, 10:22 PM

We have prepared ATAC libraries for 24 samples following the protocol from Buenrostro et.al, using their custom Nextera-like primers and we have attempted to sequence these on NextSeq.
We're essentially getting no reads and can't figure out why. The libraries show good fragment distribution on Tapestation, and we have also run a library quantification qPCR which also shows that there is sufficient sequenceable material.
We're completely stuck, and Illumina as well as the ATAC forum have been of little help...
If anyone can help, it would be very much appreciated!

pmiguel · 10-04-2017, 09:37 AM

No reads, or no clusters?
Nextera sequencing primers aren't included in HiSeq High Output chemstry reagents. But they are in MiSeq/HiSeq Rapid chemsitry and so should be in the NextSeq reagent kits.
So my next thought would be that your adapters have an error in them that is preventing any sequence from being generated. You should go to the oligonucleotide synthesis company's specification sheet for your oligos and verify the sequence provided is the Nextera adapter sequence.
--
Phillip

RickC7 · 10-04-2017, 12:35 PM

We have successfully sequenced ATAC-Seq libraries on the Next-Seq. I need to look back at my notes, but we did not use the custom primers. Loosely based off Pamela Milani (Fraenkel Lab) We used the Nextera DNA Library Kit and Nextera Index primers. Sequencing run worked fine. Did you use PhiX on the run? That would give you some indication...

flutterfly28 · 04-01-2019, 10:00 AM

Hey favwiz, did you ever figure out what was wrong?
I seem to be having the same problem with my ATAC-Seq run...

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10-03-2017, 10:22 PM	#1
favwiz Junior Member Location: London Join Date: Nov 2011 Posts: 3	Can't sequence ATAC-Seq libraries We have prepared ATAC libraries for 24 samples following the protocol from Buenrostro et.al, using their custom Nextera-like primers and we have attempted to sequence these on NextSeq. We're essentially getting no reads and can't figure out why. The libraries show good fragment distribution on Tapestation, and we have also run a library quantification qPCR which also shows that there is sufficient sequenceable material. We're completely stuck, and Illumina as well as the ATAC forum have been of little help... If anyone can help, it would be very much appreciated!

10-04-2017, 09:37 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,315	No reads, or no clusters? Nextera sequencing primers aren't included in HiSeq High Output chemstry reagents. But they are in MiSeq/HiSeq Rapid chemsitry and so should be in the NextSeq reagent kits. So my next thought would be that your adapters have an error in them that is preventing any sequence from being generated. You should go to the oligonucleotide synthesis company's specification sheet for your oligos and verify the sequence provided is the Nextera adapter sequence. -- Phillip

10-04-2017, 12:35 PM	#3
RickC7 Member Location: Baton Rouge, Louisiana Join Date: Feb 2010 Posts: 31	We have successfully sequenced ATAC-Seq libraries on the Next-Seq. I need to look back at my notes, but we did not use the custom primers. Loosely based off Pamela Milani (Fraenkel Lab) We used the Nextera DNA Library Kit and Nextera Index primers. Sequencing run worked fine. Did you use PhiX on the run? That would give you some indication...

04-01-2019, 10:00 AM	#4
flutterfly28 Junior Member Location: San Francisco Join Date: Jan 2019 Posts: 1	Hey favwiz, did you ever figure out what was wrong? I seem to be having the same problem with my ATAC-Seq run...