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Old 10-03-2017, 11:22 PM   #1
favwiz
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Location: London

Join Date: Nov 2011
Posts: 3
Exclamation 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!
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Old 10-04-2017, 10:37 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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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.
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Phillip
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Old 10-04-2017, 01:35 PM   #3
RickC7
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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...
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