PCR-free; absolute abundance; yeast

fadss · 07-08-2014, 01:01 PM

I am new to NGS.
Goal is to find absolute abundance of yeast species in a mixed sample.
I plan to use PCR-free methods since PCR could cause amplification biased.

Plan is to use DNA fragmentase and size select ~300-400bp on Gel-> clean up->
end repair->A tail-> ligation/barcode/adaptor-> MiSeq 250 pair end.

From this millions of seq.(from different part of DNA) to figured out species and it's relative abundance??? Feels like i am missing some info. somewhere.
Any potential biased or problems?

above all this i do open for other complete new suggestions....

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07-08-2014, 01:01 PM	#1
fadss Junior Member Location: Britsh Columbia Join Date: Jan 2014 Posts: 4	PCR-free; absolute abundance; yeast I am new to NGS. Goal is to find absolute abundance of yeast species in a mixed sample. I plan to use PCR-free methods since PCR could cause amplification biased. Plan is to use DNA fragmentase and size select ~300-400bp on Gel-> clean up-> end repair->A tail-> ligation/barcode/adaptor-> MiSeq 250 pair end. From this millions of seq.(from different part of DNA) to figured out species and it's relative abundance??? Feels like i am missing some info. somewhere. Any potential biased or problems? above all this i do open for other complete new suggestions....