WGS on a tiny invertebrate: what library prep and assembly strategy would you use?

[kmkocot]

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

[wbsimey]

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