SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
"allele balance ratio" and "quality by depth" in VCF files efoss Bioinformatics 2 10-25-2011 12:13 PM
Relatively large proportion of "LOWDATA", "FAIL" of FPKM_status running cufflink ruben6um Bioinformatics 3 10-12-2011 01:39 AM
The position file formats ".clocs" and "_pos.txt"? Ist there any difference? elgor Illumina/Solexa 0 06-27-2011 08:55 AM
"Systems biology and administration" & "Genome generation: no engineering allowed" seb567 Bioinformatics 0 05-25-2010 01:19 PM
SEQanswers second "publication": "How to map billions of short reads onto genomes" ECO Literature Watch 0 06-30-2009 12:49 AM

Reply
 
Thread Tools
Old 07-30-2010, 10:13 AM   #1
DoubleA
Member
 
Location: Los Angeles

Join Date: Jul 2010
Posts: 16
Default "rapid" vs "general" MID adapters

Hello Everyone,

I'm interested in producing 4 "rapid" libraries from human genomic DNA and annealing separate MID adapters to each. Instead of ordering the Rapid Library MID Adapter Kit from Roche (includes 12 different MID adapters) I would like to order 4 from IDT. I called IDT technical support and they told me each order contains two oligos to make one adapter. From looking at the Roche technical bulletin (004-2009, "general" library), my understanding is that each library requires a separate MID Adapter A and a common MID Adapter B (4 oligos in total). The technical representative from IDT told me that the same "rapid" MID adapter could be ligated to the end of each genomic DNA fragment.

In the long run I would like to use a sequence capture array to enrich each library followed by running each library together.

1) Is the technical representative from IDT mistaken?
2)Does anybody know the difference (sequence) between the "general" and "rapid" MID adapters.
3)Can I use the long oligo from each adapter for LM-PCR to increase the library yield pre- and post- sequence capture?

Thanks for your help!
Double A
DoubleA is offline   Reply With Quote
Old 07-30-2010, 11:43 AM   #2
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,135
Default

Quote:
1) Is the technical representative from IDT mistaken?
No. The new Rapid library preparation method apparently works like the Illumina library prep method. A single partially duplexed ("Y") adapter is used which is ligated to both ends of an end-polished, dsDNA fragment. Asymmetric ends are produced via the PCR primers used to amplify the library. The Rapid Library MID adapters will introduce the MID tag at both ends of the insert.
Quote:
2)Does anybody know the difference (sequence) between the "general" and "rapid" MID adapters.
If anybody does, they're not saying. Unlike with the previous library preparation protocol Roche is not publishing the Rapid prep oligo sequences. Perhaps they have shared the sequences with IDT to allow them to synthesize custom adapters.
Quote:
3)Can I use the long oligo from each adapter for LM-PCR to increase the library yield pre- and post- sequence capture?
Sorry, no idea on this one.
kmcarr is offline   Reply With Quote
Old 08-03-2010, 05:33 AM   #3
Tom Haltern
Junior Member
 
Location: Germany

Join Date: Feb 2010
Posts: 7
Default IDT adaptors

Dear kmcarr,
is IDT offering the old titanium adaptors (not Y shaped) with a T overhang in order to be compatible with the NimbleGen capture?
I want to switch to the RL prep protocol and that is the idea of my workaround.
Thanks,
Tom
Tom Haltern is offline   Reply With Quote
Old 08-03-2010, 06:28 AM   #4
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,135
Default

Quote:
Originally Posted by Tom Haltern View Post
Dear kmcarr,
is IDT offering the old titanium adaptors (not Y shaped) with a T overhang in order to be compatible with the NimbleGen capture?
I want to switch to the RL prep protocol and that is the idea of my workaround.
Thanks,
Tom
I can't speak for IDT; you should consult with them directly.
kmcarr is offline   Reply With Quote
Old 08-03-2010, 01:37 PM   #5
Baseless
Member
 
Location: Germany

Join Date: Feb 2010
Posts: 32
Default

Since there seem to be more people sharing my pain, let me share what we tried, maybe it leads to a solution.

We, too, are into SeqCap arrays. And since manpower is hard to come by, we would greatly love to adapt to automated library prep. What we got was the Beckman SPRI-TE since it can handle both platforms we have - fortunately for most of the community, Beckman adapted to the rapid protocol pretty fast, unfortunately for us SeqCap + Rapid is not recommended at this time.

So for once, I thought I was smart, grabbed the Roche Titanium MID TCBs, added a T overhang , moved the PTO modifications to the last four bases and gave it a try. What I got was a Bioanalyzer trace with a real sweet size distribution.
The sun was shining, life was great.

Then I tried to amplify as I did with my handmade-libraries using the LM-PCR primers from the Titanium optimized SeqCap protocol. They should produce a PCR product from first base of adaptor A till last base of adaptor B. They do very robust on hand made libraries - but not on my robotic ones with the modified adaptors.

My first guess was an aberrant oligo charge or a failed annealing of the adaptors, but I ruled out both with a new annealing and new adaptors from a diffrent oligo manufacturer.

So now I still have something that looks like a nice library on the Bioanalyzer trace, but which refuses to amplify.

While I thought that 'just adding the overhang cant be too hard' I am running out of ideas lately...Anyone solved this riddle in their lab yet?
Baseless 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 10:06 PM.


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