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Old 02-08-2010, 03:22 PM   #1
PaulineF
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Default SE adapters and PE primers

As dumb as this sounds, can someone please tell me if PE primers can work with SE adapters to successfully generate PE data? We suspect this combination may have occurred on 3 occasions but each time there was a different outcome: only read 1 worked (GAII, long time ago), both reads generated clusters and went through the pipeline without errors, and no clusters at all (latter 2 on GAIIx, SCS2.5).
I checked some helpful diagrams provided by this group and it looks like it should not have worked at all. But I cannot explain our 3 different results, so any help will be greatly appreciated.
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Old 02-09-2010, 10:53 AM   #2
Michael.James.Clark
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I do not think it should work because the sequences of the PE adapters are different from the SE adapters if I remember correctly.
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Old 02-09-2010, 11:10 AM   #3
PaulineF
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Yes, exactly, the SE and PE adapters and primer 1's are different. However, after taking a closer look, because the the PE1 primer differs from the SE1 primer only by having that insert, so PE1 could potentially hybridize to the SE adapter by looping out the insert. Once that 1st strand is amplified, then SE2 and PE2 are the same so the PCR can continue. This would be consistent with the lower than expected cluster densities with the suspected run that did generate both read1 and read2 data. Although, even I am not convinced that this explains our results. If hybridization and PCR efficiency are both by chance, it's pretty amazing that we got consistent results between each batch of samples.
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