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Old 01-23-2011, 12:24 PM   #1
Sophie Rose
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Question 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 05:40 PM. Reason: misspelling
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Old 01-24-2011, 05:47 AM   #2
pmiguel
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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.

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Old 01-24-2011, 09:58 AM   #3
krobison
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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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