WGS prep of micro Eukaryotes

wbsimey · 08-26-2016, 02:21 PM

We are planning to sequence a denovo reference genome of a micro Eukaryote, but cannot extract enough DNA from a single individual for PacBio or Nextera Illumina protocols. We have extracted DNA from 68 individuals of a single population and get 3.56ng total (100ul of 35.6pg/ul). We plan to pool these individuals for a "composite" genome. Our concerns include: 1) are they all the same species/lineage? 2) how useful is a ref genome generated from a composite of individuals? and 3) what will the assembly challenges be with the likely high rate of variation in reads?
We have not seen many publications on this topic. Is anyone aware of publications on such a project or have any thoughts on this?
thanks,

luc · 08-26-2016, 04:21 PM

Hi wbsimey,

as mentioned in the other post I also would recommend to work with a single cell in this case (I assume a single individual can not propagate) and amplify the sample by whole genome amplification before library prep (MDA).
Fro prokaryotes this has been done frequently.

wbsimey · 08-27-2016, 10:32 AM

Quote:

Originally Posted by luc

Hi wbsimey,

as mentioned in the other post I also would recommend to work with a single cell in this case (I assume a single individual can not propagate) and amplify the sample by whole genome amplification before library prep (MDA).
Fro prokaryotes this has been done frequently.

Thank you Luc. When you say "as mentioned in the other post ..." which post are you referring to? Do you have a reference/protocol for whole genome amplification? Can this be done on a multicellular organism rather than a single cell?

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08-26-2016, 02:21 PM	#1
wbsimey Member Location: san francisco Join Date: Jul 2010 Posts: 16	WGS prep of micro Eukaryotes We are planning to sequence a denovo reference genome of a micro Eukaryote, but cannot extract enough DNA from a single individual for PacBio or Nextera Illumina protocols. We have extracted DNA from 68 individuals of a single population and get 3.56ng total (100ul of 35.6pg/ul). We plan to pool these individuals for a "composite" genome. Our concerns include: 1) are they all the same species/lineage? 2) how useful is a ref genome generated from a composite of individuals? and 3) what will the assembly challenges be with the likely high rate of variation in reads? We have not seen many publications on this topic. Is anyone aware of publications on such a project or have any thoughts on this? thanks,

08-26-2016, 04:21 PM	#2
luc Senior Member Location: US Join Date: Dec 2010 Posts: 468	Hi wbsimey, as mentioned in the other post I also would recommend to work with a single cell in this case (I assume a single individual can not propagate) and amplify the sample by whole genome amplification before library prep (MDA). Fro prokaryotes this has been done frequently.