SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
BWA and gaps efoss Bioinformatics 5 04-15-2013 08:42 PM
What is "25 mer probes at 38 bp resolution"? tfcheng General 2 03-22-2011 07:06 AM
Nimblegen Probes aleferna Bioinformatics 1 08-10-2010 01:25 PM
How many gaps??? saima Bioinformatics 6 04-28-2010 07:33 PM

Reply
 
Thread Tools
Old 04-30-2013, 04:33 PM   #1
seraphin
Junior Member
 
Location: NY

Join Date: Feb 2013
Posts: 4
Default isses about gaps in the probes from NimbleDesign

We aim to use Nimblegen seqcap EZ choice library to target-sequence ~ 100 genes of interests including promoters, coding and intronic regions. NimbleDesign tool provides the information of the probes used for target enrichment. However, when I visualize the probe coverage under UCSC genome browser, several genes contain exons that will NOT be covered by the probes (i.e. gaps).

If I understand the principles of target enrichment correctly, as the sole purpose of the probes is to "enrich" the chromosome regions of interest and all chromosomes are first shattered to be ~500 bp to 1kb in length. Is it correct to assume that if the "uncovered" exons are only a few hundred bases away from the probes, the exons will still be enriched and we'll get the sequence of them.

Any insight to share will be greatly appreciated. Thank you
seraphin is offline   Reply With Quote
Old 04-30-2013, 04:45 PM   #2
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,355
Default

Depends on your library insert size...and the GC content of the region...most protocols recommend shorter inserts than what you've said.

The coverage profile around a given probe "island" will depend most highly on the insert size. If it's a critical target, I would figure out WHY they can't design probes (repeat elements, GC content, etc)...and perhaps there is some custom design that you could do to get the region.
ECO is offline   Reply With Quote
Old 04-30-2013, 05:06 PM   #3
seraphin
Junior Member
 
Location: NY

Join Date: Feb 2013
Posts: 4
Default

Any references/documents explaining why most protocols "recommend" shorter inserts, or is it just because most sequencing machines these days prefer to sequence 50-150 base DNA pieces? It's a surprise to us too that the "exons" would not be covered, as I thought Nimblegen exome seq kits have been quite popular.
ps. I actually tried to contact their tech support, but recently Roche is restructuring their Diagnostics division (esp Sequencing group) and somehow I could not get a hold of their tech support. Anyone else with a similar experience these days?
seraphin is offline   Reply With Quote
Old 05-01-2013, 03:27 AM   #4
Zaag
Senior Member
 
Location: Amsterdam

Join Date: Nov 2009
Posts: 112
Default

Usually the relatively small insert size is because of the clonal amplification step before sequencing.

You do see that probes in the neighborhood of you target help getting the target covered. However this can be difficult with short read platforms.

About roche/nimblegen tech "support"... Well let's say if you have a small target (< 1 Mb) it might be faster to do sangersequencing than to wait for them to answer any question in a meaningful way. If you have a bigger target you might want to consider whole genome More serious: horrible company with good products.
Zaag is offline   Reply With Quote
Reply

Tags
nimblegen, target capture

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 03:40 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO