SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
HiSeq insert size njlodato Sample Prep / Library Generation 0 09-04-2015 10:03 AM
Maximum insert size with Hiseq 3000 upendra_35 Illumina/Solexa 6 07-23-2015 03:44 AM
What insert size for velveth (1.2.10) with 2 sets of reads with diff. insert sizes? Genomics101 Bioinformatics 4 02-07-2014 11:41 AM
150bp-1.3kb insert size PE on HiSeq jmugford Illumina/Solexa 5 04-19-2012 07:43 PM
About Insert, Insert size and MIRA mates.file aarthi.talla 454 Pyrosequencing 1 08-01-2011 01:37 PM

Reply
 
Thread Tools
Old 11-04-2016, 05:26 AM   #1
JBKri
Member
 
Location: Heraklion, Greece

Join Date: Jan 2014
Posts: 77
Default HiSeq X different insert size?

I am about to prepare TruSeq PCR-free libraries for HiSeq X sequencing.
From:
http://support.illumina.com/sequenci...questions.html

"The supported insert size on the HiSeq X system is 350 bp and 450 bp for TruSeq Nano DNA and TruSeq DNA PCR-Free libraries."

As I see it this phrase could be interpreted in two ways; 350 bp for Nano and 450 bp for PCR-free, or both types of libraries can use either insert size. Anyone knows which it is? If the first; does that mean we MUST make our insert size 450 bp? There is not even a protocol for that in the TruSeq manual.

Jon
JBKri is offline   Reply With Quote
Old 11-04-2016, 05:47 AM   #2
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,077
Default

HiSeq X uses ordered flowcells (like HiSeq 3000/4000). There is a need to control insert size (between 350-450 bp, ideally) with ordered flowcells to avoid generation of optical duplicates due to pad hopping (DNA fragments contaminating multiple wells nearby during the ExAmp procedure).

While you could load longer libraries, you would need to deal with the optical duplicate issue/loss of data downstream.
GenoMax is offline   Reply With Quote
Old 11-04-2016, 06:10 AM   #3
JBKri
Member
 
Location: Heraklion, Greece

Join Date: Jan 2014
Posts: 77
Default

Quote:
Originally Posted by GenoMax View Post
HiSeq X uses ordered flowcells (like HiSeq 3000/4000). There is a need to control insert size (between 350-450 bp, ideally) with ordered flowcells to avoid generation of optical duplicates due to pad hopping (DNA fragments contaminating multiple wells nearby during the ExAmp procedure).

While you could load longer libraries, you would need to deal with the optical duplicate issue/loss of data downstream.
Thanks. Does this mean we should do a more stringent size selection than the standard TruSeq two-sided magnetic bead size selection? Do you have any links to protocols for this? The Illumina Custom Protocol selector seems to be broken at the moment.
JBKri is offline   Reply With Quote
Old 11-04-2016, 07:01 AM   #4
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,077
Default

I will let someone else answer that since it is about experimental protocol.
GenoMax is offline   Reply With Quote
Old 11-04-2016, 05:25 PM   #5
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 451
Default

The HiSeq 4000, that we are running, is basically the same sequencer as the X sequencer using the same chemistry. Bead based size selection is perfectly sufficient over here.

(if anybody has seen any evidence that pad hooping actually occurs more frequently for longer insert libraries I would be interested).

Last edited by luc; 11-04-2016 at 05:29 PM.
luc is offline   Reply With Quote
Old 11-05-2016, 06:42 AM   #6
JBKri
Member
 
Location: Heraklion, Greece

Join Date: Jan 2014
Posts: 77
Default

Quote:
Originally Posted by luc View Post
The HiSeq 4000, that we are running, is basically the same sequencer as the X sequencer using the same chemistry. Bead based size selection is perfectly sufficient over here.
..

Great, that's just what I was hoping for!
JBKri is offline   Reply With Quote
Reply

Tags
hiseq x, insert size, truseq pcr-free

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 04:07 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO