Sanger Sequencing for Community Analysis

LMM · 04-16-2018, 12:32 PM

Hello
I have environmental cDNA left over from a tricky qPCR project. Is it possible to use Sanger Sequencing of the ITS 5.8S rDNA region to observe the community at even the order level?

gringer · 04-17-2018, 05:32 PM

Unlikely. Sanger sequencing doesn't really work with variants at a frequency below about 20%, and any INDEL variation in the community is going to mess up the sequencing quite a lot.

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04-16-2018, 12:32 PM	#1
LMM Junior Member Location: Athens, Ohio Join Date: Jan 2017 Posts: 8	Sanger Sequencing for Community Analysis Hello I have environmental cDNA left over from a tricky qPCR project. Is it possible to use Sanger Sequencing of the ITS 5.8S rDNA region to observe the community at even the order level?

04-17-2018, 05:32 PM	#2
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 838	Unlikely. Sanger sequencing doesn't really work with variants at a frequency below about 20%, and any INDEL variation in the community is going to mess up the sequencing quite a lot.