SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
ATAC seq lysis time zoe561 Sample Prep / Library Generation 3 03-05-2015 09:00 AM
Error in RNA-seq analysis of Arabidopsis following how-to- seq wiki protocol himanshu04 RNA Sequencing 1 03-23-2012 06:58 AM
Anyone has protocol for Ion PES protocol? marcowanger Ion Torrent 1 01-18-2012 08:28 PM
illumina alternative v1.5 protocol for small rna seq vs. the standard protocol ik76 Sample Prep / Library Generation 1 03-25-2010 02:24 PM
mRNA-Seq protocol Xi Wang Illumina/Solexa 5 11-05-2009 06:41 AM

Reply
 
Thread Tools
Old 06-07-2017, 08:59 AM   #61
mcnaircm
Junior Member
 
Location: Philadelphia, PA

Join Date: May 2017
Posts: 2
Default

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
mcnaircm is offline   Reply With Quote
Old 06-07-2017, 01:08 PM   #62
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,218
Default

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.

--
Phillip
pmiguel is offline   Reply With Quote
Old 06-07-2017, 04:29 PM   #63
mcnaircm
Junior Member
 
Location: Philadelphia, PA

Join Date: May 2017
Posts: 2
Default

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.
mcnaircm is offline   Reply With Quote
Old 06-07-2017, 04:52 PM   #64
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 1,035
Default

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.
nucacidhunter is offline   Reply With Quote
Old 08-11-2017, 06:34 AM   #65
jamesdinn
Junior Member
 
Location: Copenhagen

Join Date: Dec 2016
Posts: 2
Default

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
jamesdinn is offline   Reply With Quote
Old 08-11-2017, 06:52 PM   #66
finixtree
Junior Member
 
Location: NC

Join Date: Aug 2015
Posts: 2
Default

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
finixtree is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 02:16 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO