SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Mate pair libraries with longer read length rakesh.ponnala Illumina/Solexa 4 01-16-2012 02:41 PM
exome enriched mate-pair libraries upenn_ngs Illumina/Solexa 0 07-29-2011 09:26 AM
Use of illumina mate pair libraries for scaffolding glacerda Bioinformatics 2 07-13-2011 07:35 AM
Sequencing Illumina mate pair libraries beyond 36bp PNG Sample Prep / Library Generation 2 08-24-2010 11:54 AM
Difference between mate pair and pair end bassu General 2 06-19-2010 06:13 AM

Reply
 
Thread Tools
Old 01-23-2011, 11:46 PM   #1
Daytwa
Member
 
Location: Detroit

Join Date: Dec 2010
Posts: 19
Default Very Large Mate Pair Libraries

I am unclear on why the mate pair library protocol requires gap sizes to be under 5 kb. Is this just due to the limits of column purification or reliable fragmentation?

Has anyone ever tried to prep a library using larger gap sizes?

Thanks.
Daytwa is offline   Reply With Quote
Old 01-24-2011, 02:46 AM   #2
volks
Member
 
Location: hd.de

Join Date: Jun 2010
Posts: 81
Default

probably the circularization step becomes less efficient with larger gap sizes.
volks is offline   Reply With Quote
Old 01-24-2011, 04:58 AM   #3
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 506
Default

Three addition considerations:
1) the molar concentration of your library decreases as the size increases
2) size selection is less accurate (i.e., the range is broader) as the size increases
3) larger gap sizes will skip smaller contigs, increasing the errors in assembly
HESmith is offline   Reply With Quote
Old 01-24-2011, 06:59 AM   #4
Daytwa
Member
 
Location: Detroit

Join Date: Dec 2010
Posts: 19
Default

Thanks for the responses so far.

Any idea why the Solid and Solexa protocols differ in regards to how they open the circularized product (Restriction enzyme vs shearing)?
Daytwa is offline   Reply With Quote
Old 01-24-2011, 07:20 AM   #5
volks
Member
 
Location: hd.de

Join Date: Jun 2010
Posts: 81
Default

the solid protocol now uses nick translation to allow for reads longer than 2x25bp as permitted by the EcoP15I restriction enzyme.
the advantage compared to (random) shearing is that the internal adapter is more likely to be located in the middle of the product, and is less likely to be sequenced from either end. but i am no expert on the illumina protocol.

Last edited by volks; 01-24-2011 at 07:27 AM.
volks is offline   Reply With Quote
Old 01-24-2011, 07:25 AM   #6
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 506
Default

Shearing is random (in theory), while the restriction digest strategy yields a defined read length (25bp) from each end.
HESmith is offline   Reply With Quote
Old 01-25-2011, 12:27 PM   #7
JakobHedegaard
Member
 
Location: Aarhus, Denmark

Join Date: Mar 2008
Posts: 57
Default

We have prepared ~10 Kbp mate-pair libraries using Illuminas v2 kit. But with lower diversity than a smaller insert size library.
It would be nice if an adapter sequence could be included between the two ends facilitating the detection of the "break-point" and thereby reads longer than the 36 bp as recommended by Illumina to avoid (or at least minimize...) chimeric sequences.
Cheers,
Jakob
JakobHedegaard is offline   Reply With Quote
Old 04-21-2015, 01:37 AM   #8
riham
Junior Member
 
Location: usa

Join Date: Apr 2015
Posts: 1
Default

You can join us for best cert killer PCNSE6 dumps oracle training solutions.

Last edited by riham; 06-18-2015 at 12:28 AM.
riham 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 08:36 AM.


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