SOLiD PE Chemistry

Sophie Rose · 01-23-2011, 01:24 PM

Hello, we've been trying out the PE 50/35 sequencing chemistry and the reverse (35bp) read quality is poor. Does anyone know which step(s) of the 35bp chemistry makes it less efficient than the forward reaction? Is it due to the 5'->3' ligase or an aspect of the extra capping/dephosphorylation steps?

pmiguel · 01-24-2011, 06:47 AM

My guess is that it is the latter.
But another factor is that Lifetech has not been tinkering with the chemistry they use to do 5' extensions as they have the 3' extensions. I hope this means there will be improvement as that chemistry is optimized. Currently, when all goes well, the 5' extension chemistry looks to me about like things did under v2 3' chemistry.

--
Phillip

krobison · 01-24-2011, 10:58 AM

I've also seen poorer quality on the reverse read.

Despite a few people trying to educate me, I'm still confused why the reverse reads are more difficult. But, it's very hard to see how the ligation step could be the issue -- ligase shouldn't know which fragment you think is front and which you think is back, it's just looking to make a join between two fragments.

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01-23-2011, 01:24 PM	#1
Sophie Rose Junior Member Location: California Join Date: Nov 2010 Posts: 1	SOLiD PE Chemistry Hello, we've been trying out the PE 50/35 sequencing chemistry and the reverse (35bp) read quality is poor. Does anyone know which step(s) of the 35bp chemistry makes it less efficient than the forward reaction? Is it due to the 5'->3' ligase or an aspect of the extra capping/dephosphorylation steps? Last edited by Sophie Rose; 01-23-2011 at 06:40 PM. Reason: misspelling

01-24-2011, 06:47 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	My guess is that it is the latter. But another factor is that Lifetech has not been tinkering with the chemistry they use to do 5' extensions as they have the 3' extensions. I hope this means there will be improvement as that chemistry is optimized. Currently, when all goes well, the 5' extension chemistry looks to me about like things did under v2 3' chemistry. -- Phillip

01-24-2011, 10:58 AM	#3
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 747	I've also seen poorer quality on the reverse read. Despite a few people trying to educate me, I'm still confused why the reverse reads are more difficult. But, it's very hard to see how the ligation step could be the issue -- ligase shouldn't know which fragment you think is front and which you think is back, it's just looking to make a join between two fragments.