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

Similar Threads
Thread Thread Starter Forum Replies Last Post
Plasmid library sequencing-which platform (if any)? zaratieg General 4 01-22-2013 11:01 AM
sequencing cost for Nexera library v.s. Truseq? kanfenfen Core Facilities 11 08-27-2012 02:08 PM
Sequencing Library Construction of Solexa chloe_giselle Illumina/Solexa 2 12-11-2011 11:56 AM
miRNA library construction/sequencing lvcosme General 0 09-20-2011 01:30 PM
Sequencing smRNA library from opposite end? Goodluck Illumina/Solexa 0 09-09-2009 01:45 PM

Thread Tools
Old 05-10-2013, 11:01 AM   #1
Junior Member
Location: LA

Join Date: May 2013
Posts: 1
Default Library sequencing

I am planning on performing an illumina multiplex sequencing run on a library of sequences that I have compiled. They are digested out of plasmid and consist of ~10 unvaried nucleotides, 60 varied nucleotides, ~40 unvaried nucleotides, 60 varied nucleotides, then 10 unvaried nucleotides in that order. I have seen in previous papers that there are some issues with the cluster identification step for sequences which are unvaried in their first few nucleotides. Is this still a major problem? Is there a way to get around this without adding extra varied nucleotides to the ends of my sequences? Or is this the only robust solution?
sleeplessstudent is offline   Reply With Quote
Old 05-10-2013, 12:23 PM   #2
Senior Member
Location: Boston,MA

Join Date: Nov 2008
Posts: 122

You need to design a custom sequencing primer.

Use the sequence of the adapter+sequence of your non-variable region(10 bases)

Then create a sequencing primer that is complementary to the last base of your nonvariable region-then work back.

When the Tm of the primer is above 65 (that way it will work on both HiSeq and MiSeq) you have that primer synthesized and HPLC purified.

Use the primer at 100uM.

Whoever is doing you sequencing will know how to use it.
kwaraska is offline   Reply With Quote
Old 05-10-2013, 12:51 PM   #3
Registered Vendor
Location: Eugene, OR

Join Date: May 2013
Posts: 521

If you are sequencing a small number of plasmid-derived inserts, then you are likely to not need the full number of reads in a lane. An alternative to creating a custom primer is to just spike your library into a control lane of PhiX on a HiSeq, or mix in a higher-complexity library (usually someone has something they want a little more sequence for) on a MiSeq. It seems like the recent software changes have made the MiSeq less sensitive to complexity reductions anyway.
SNPsaurus is offline   Reply With Quote
Old 05-13-2013, 11:00 PM   #4
Junior Member
Location: Pretoria

Join Date: May 2013
Posts: 1

Please can your help in any library for plasmodium gametocytes. Thnaks . Erny
tambo is offline   Reply With Quote

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 12:19 AM.

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