SEQanswers

Go Back   SEQanswers > General



Similar Threads
Thread Thread Starter Forum Replies Last Post
'Assembly PCR' for homemade amplicon library prep esherman Illumina/Solexa 18 11-22-2014 03:28 PM
Cheapest 96 well WGS sample prep andibody Sample Prep / Library Generation 3 02-18-2013 12:49 AM
Strategy for genome assembly mbseq Bioinformatics 5 10-25-2012 06:04 AM
how to optimize viral genome assembly in WGS 7.0? rexxi Bioinformatics 1 08-01-2012 11:01 PM
De novo assembly strategy Wiseone De novo discovery 0 11-18-2010 08:30 AM

Reply
 
Thread Tools
Old 05-20-2015, 08:17 PM   #1
kmkocot
Member
 
Location: Alabama

Join Date: Jun 2009
Posts: 48
Default WGS on a tiny invertebrate: what library prep and assembly strategy would you use?

Hi all,

We are planning to sequence the genome of a tiny marine invertebrate. DNA extraction from a single individual will likely yield only around 10 ng of DNA and we only want to use one individual to help reduce issues with heterozygosity.

Our current plan is to use the Qiagen RepliG Mini kit on one starved individual, make Illumina PCR-free sequencing libraries with an insert of around 450 bp, and sequence that library to >60X coverage with 2 X 250 bp PE on a HiSeq 2000 (following the recommendations for Discovar de novo, which we plan to use to assemble the genome).

We will also do a Clontech SMARTer transcriptome on a second starved individual for gene model annotation purposes.

Would you do it differently? I know chimaerism can be a problem with RepliG but since we will be fragmenting the DNA we expect few pairs of reads to span a chimaeric break.

Also, does anyone have experience with using RepliG-amplified DNA for Illumina mate pair libraries? I'm guessing we could alternatively make libraries compatible with allpaths but keep our mate pair library insert size below 3 kb or so (to help avoid chimaeric breaks)

Thanks!
Kevin
kmkocot is offline   Reply With Quote
Old 08-26-2016, 02:06 PM   #2
wbsimey
Member
 
Location: san francisco

Join Date: Jul 2010
Posts: 11
Default Micro organism

Hello kmkocot,
we too are planning to sequence a ref genome for a micro eukaryote. Did you ever go forward with your tint invert? and if so, did you use a single individual or did you pool multiple individuals?
thanks,
Brian
wbsimey 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 11:05 AM.


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