SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
clustering algorithm for single reads from transposon integrations Retro Bioinformatics 3 02-21-2012 05:58 PM
Illumina to sequence region of 50 kB Marius Illumina/Solexa 4 08-15-2011 02:05 PM
PubMed: Sequence Capture and Next Generation Resequencing of the MHC Region Highlight Newsbot! Literature Watch 0 05-31-2011 02:00 AM
attempting to sequence human MHC region DoubleA Genomic Resequencing 6 11-14-2010 08:40 PM
Vector trimming: are flanking sequences sufficient? sulicon Bioinformatics 1 09-20-2010 07:02 AM

Reply
 
Thread Tools
Old 01-12-2012, 02:39 AM   #1
Akira
Member
 
Location: Malaysia

Join Date: Sep 2010
Posts: 16
Question Sequence transposon flanking region

I am new to NGS first of all. I would like to sequence transposon flanking region, thus during PCR amplification step, instead of using primers supplied by Illumina TruSeq, I will use own customized primer (tailed with Illumina adapter sequence) which will bind specifically to the transposon end and start amplification. Lots of issue I need to settle for sample prep. But now my doubt is: during sequencing run, is it possible if we use own sequencing primer (in which I plan to design primer which binds to the transposon end region)? Say if I wanted to run on HiSeq2000. If I use standard sequencing primer, most of the first ~20bp of the reads will be the same (the transposon end) and how will this affect the coordination of clusters during first few cycles?

Sorry for the poor English used.
Akira is offline   Reply With Quote
Old 01-12-2012, 04:14 AM   #2
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 506
Default

Low complexity sequence during the first four cycles will adversely affect cluster calling. Sequencing with a custom primer is fine, as long as the Tm is comparable to the standard sequencing primer.
HESmith is offline   Reply With Quote
Old 01-12-2012, 05:46 AM   #3
Akira
Member
 
Location: Malaysia

Join Date: Sep 2010
Posts: 16
Default

Many thanks to the reply.

If paired end, will the cluster calling be repeated for read 2? Or once the first four cycles of read 1 have coordinated the clusters position, it doesn't matter if read 2 starts with low complexity sequence? For sure more than 10bp of the first read will be low complexity sequence (as mentioned the transposon end), I was thinking if I design the primer in a way the transposon end is actually located on read 2, will it still affecting the cluster coordination?

Custom sequencing primer can replace the original primer? I have been told that it comes in a master mix together with other reagents needed for sequencing run. Is it true?
Akira is offline   Reply With Quote
Old 01-12-2012, 05:50 AM   #4
krobison
Senior Member
 
Location: Boston area

Join Date: Nov 2007
Posts: 747
Default

By low complexity, it is meant low diversity of the sequence -- your primers may cause issues due to the fixed targeting region. One way to try to address this would be to use a pool of primers for the amplification step, with different lengths of spacer between the targeting region and the Illumina tail -- this will prevent the constant region from always being in register & should help with the cluster calling (idea is not mine; someone published a scheme in which the multiplex tags used variable lengths to solve the problem)
krobison is offline   Reply With Quote
Old 01-12-2012, 05:56 AM   #5
Akira
Member
 
Location: Malaysia

Join Date: Sep 2010
Posts: 16
Default

Many thanks for the reply. Cluster calling happens in both read 1 and read 2 for paired end sequencing or only read 1? Most of the clusters will be filtered out if the clusters calling was not good enough, am I right?
Akira is offline   Reply With Quote
Old 01-12-2012, 05:58 AM   #6
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 506
Default

Cluster calling is based solely on the first four cycles of read one, so low diversity of read two will not affect the data.

Custom primers can be used during clustering on the cBOT. Select the appropriate protocol and load the primers in tube strip. See pp. 53-4 of the cBOT user's guide for more info.
HESmith is offline   Reply With Quote
Old 01-12-2012, 06:05 AM   #7
Akira
Member
 
Location: Malaysia

Join Date: Sep 2010
Posts: 16
Default

Thanks HESmith. In this case, I can actually revert back the concept by starting to sequence from the other side of the fragments, which is the genomic DNA as read 1, then the transposon end with low sequence complexity as read 2. Am I right? Then only I filter out those sequence reads without any transposon end tag on read 2 using bioinformatic tools, possible? Because I am only interested in those reads tagged with transposon end, it doesn't matter the tag is on read 1 or read 2. I hope I get the whole picture right.

Your advice is much appreciated.
Akira is offline   Reply With Quote
Old 03-15-2012, 09:45 AM   #8
Janiz
Junior Member
 
Location: San Diego

Join Date: Mar 2012
Posts: 2
Default

Hi Akira,

I am considering using Epicentre's (now Illuminas) Nextera DNA sample prep kit. It seems there's been some changes to this kit this year and the index primers are now sold separately. Do you possibly know the sequences of the tagged DNA after the tagmentation and cleanup? Is it possible to create our own primers to measure tagmentation efficiency without having to order the index kit? In this new protocol it seems you need the index kit (at least Index 1 and 2) to be able to perform the PCR amplification step and add on the adapters (after which you could do a QPCR and measure tagmentation efficiency) although they do provide the PCR buffer and primer mix?

Thanks!
Janiz is offline   Reply With Quote
Old 03-18-2012, 06:53 AM   #9
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

Quote:
Originally Posted by HESmith View Post
Cluster calling is based solely on the first four cycles of read one, so low diversity of read two will not affect the data.
While the former point is correct, I think the latter point no longer is. Prior to a firmware/software update last year (I think the one that allowed use of v3 chemistry) the reverse PE read apparently just used the focal settings acquired during the first cycle of read1. This was good in that diversity was not an issue for this read. But it was bad if there was an index read before the reverse PE read and this index read had hosed the focal settings due to its low diversity. (Like if you had some indexed lanes and some non-indexed, or single index lanes.)

Under such circumstances the work-around was to exit HCS entirely prior to turn-around. Upon re-entering HCS, the software would force a focal settings calibration prior to read 2. So Illumina changed the software so it always does the re-calibration prior to read 2. But that leaves you with the low diversity bug for both read 1 and read 2.

At least this is my interpretation of where things stand currently. Corrections welcome, of course.

--
Phillip
pmiguel is offline   Reply With Quote
Reply

Tags
illumina ga, illumina hiseq, pcr primers, sequencing primers, transposon

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 02:38 PM.


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