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Old 06-07-2017, 08:59 AM   #61
mcnaircm
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Hi all,
I'm just piggybacking on this thread, but I think it is probably the best place to post my question - and something I can't be the only person having an issue with!
I'm prepping my samples for ATAC-Seq (planning to do single end sequencing 75bp reads, just looking at regions of open chromatin), and I know that most of the protocols have custom primers (both Ad1 and index primers). I was wondering if you HAD to use these, or if there were capable primers in the Nextera library prep kit and index kit to prep the samples. Again, I'm not sure if people use the custom primers because it is cheaper or if it is because Illumina is so vague with their primer mixes and cocktails. Any input would be appreciated!

Cheers
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Old 06-07-2017, 01:08 PM   #62
pmiguel
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Not sure what protocol you are using, but the only one I have seen seemed to use somewhat modified Nextera adapters. This means that to sequence these libraries on a HiSeq 2500 you need to either do a Rapid run (which includes nextera sequencing primers) or add the appropriate Nextera primers to your high output reagents. We would be loathe to do the latter unless the investigator was buying all 8 lanes of the flow cell.

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Old 06-07-2017, 04:29 PM   #63
mcnaircm
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Hi Phillip,
So you are correct in that the majority of the protocols that I have seen use modified Nextera adapters. My question was whether or not these modified adapters had to be used (or were used mainly because they were cost effective), or if there were already reagents in the library and index kits for the nextera prep that would work.

Additionally, adding the primers to the high output reagents wouldn't be problem, since we have indeed purchased the entire flow cell.
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Old 06-07-2017, 04:52 PM   #64
nucacidhunter
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ATAC-seq primers differ from Nextera by:
1- Lack of i5 index in Ad1 (equivalent to Nextera index2 read primer) so libraries are single indexed
2- Addition of 6-7 bases to 3 end of primers that extends primer binding into transposome recognition sequence common between both adapters

I do not see any difference beside possible different concentrations of adapters used in PCR noting that Nextera adds PPM to supplement indexed primers as their concentration is lower than needed for Nextera. If you are using Nextera primers you might need to adjust annealing temperature and primer volume to optimise reaction.
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Old 08-11-2017, 06:34 AM   #65
jamesdinn
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Hi all,
I would like to ask you about atac-seq sequencing,
I sequence a library for 100M reads, and after filtering i get much less reads (TotalReadPairs:9 thousands !!) and i don't know whay this huge difference after filtering, Filtering file:
xxx.trim.filt.srt.nodup.pbc.qc

best
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Old 08-11-2017, 06:52 PM   #66
finixtree
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What are those reads filtered out for? low quality score, unmappable, mapped to mtDNA? Need more information to give you even a guess.
Quote:
Originally Posted by jamesdinn View Post
Hi all,
I would like to ask you about atac-seq sequencing,
I sequence a library for 100M reads, and after filtering i get much less reads (TotalReadPairs:9 thousands !!) and i don't know whay this huge difference after filtering, Filtering file:
xxx.trim.filt.srt.nodup.pbc.qc

best
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Old 02-13-2018, 07:48 AM   #67
bman
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Quote:
Originally Posted by nucacidhunter View Post
ATAC-seq primers differ from Nextera by:
1- Lack of i5 index in Ad1 (equivalent to Nextera index2 read primer) so libraries are single indexed
2- Addition of 6-7 bases to 3 end of primers that extends primer binding into transposome recognition sequence common between both adapters

I do not see any difference beside possible different concentrations of adapters used in PCR noting that Nextera adds PPM to supplement indexed primers as their concentration is lower than needed for Nextera. If you are using Nextera primers you might need to adjust annealing temperature and primer volume to optimise reaction.
I'm in the same boat here. Have Nextera Index kit and was wondering if I could use it rather than the custom primers from the protocol. Anyone have any experience with this?
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Old 02-13-2018, 08:32 AM   #68
pmiguel
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Quote:
Originally Posted by bman View Post
I'm in the same boat here. Have Nextera Index kit and was wondering if I could use it rather than the custom primers from the protocol. Anyone have any experience with this?
The NexteraXT index kit could work--however the standard ATACseq protocol expects the primer concentrations to be ~5X higher than they are supplied in the NexteraXT index kits. To find this out we just measured the concentration of the index oligos (which Illumina refuses to disclose for unknown reasons) using a Qubit.

So you could speed vap the Nextera index primers, or add more to the reactions (instead of the water in the protocol) or both. Or just use less primer, I suppose. Might still work.

I would recommend just ordering dual index oligos for the PCR step from a reputable oligo synthesis company. If you are planning to run these together in the same lane of an Illumina patterned flowcell instrument (HiSeq 3000/4000/X or NovaSeq -- and now maybe iSeq?) then you would want to use unique dual indexes anyway...

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Old 02-13-2018, 08:45 AM   #69
bman
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Phillip,
Thank you very much for the help. I think that ordering the custom primers for the future is definitely something that we will do, but I have the nextera index kit so was hoping to use it. I have found several examples of protocols that claim to have used the nextera index kit but clearly most people use the custom primers. I will try the nextera kit out and see what kind of results I get.
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Old 02-22-2018, 08:01 AM   #70
bman
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I have been working on optimizing an atac-seq protocol on frozen tissue. I have been trying a few different things, and have attached bioA traces from two of my best runs. Has anyone ever gotten results like this? If so, is it sequenceable? I have found it hard to find a consensus "perfect" bioA trace for an atac prep online. I feel like the library might be best with some more input material. I also can't figure out why my 190 bp peak is consistently smaller than the 300 bp peak.





Thanks!

Last edited by bman; 02-22-2018 at 09:36 AM.
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