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Old 05-07-2010, 07:28 AM   #1
lzembek
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Default Linker Bias in 454 Paired-End Libraries

In 454 paired-end sequencing analysis (3, 8, or 20kb), has anyone found that the sequence on the left of the linker to be significantly shorter than the sequence on the right? Theoretically, a randomly sheared sample should generate reads with sequences of equal length, on average, on either side of the linker. However, I am seeing a bias for the linker to be located near the start of the read. Could the circular products be more prone to shearing at a site closer to the left side of the linker?
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Old 05-18-2010, 12:43 AM   #2
flxlex
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Your post made me check, but both for the two smaller datasets (bacterial genome 3kb old GS FLX and Titanium 8kb PE), and the larger one (eukaryotic genome Titanium 3kb, 8kb, 20kb) I don't see a significant difference between the length distributions of the left half and the right half.

Also, when I plot the position of the linker (I take the start position of the right half from the 454TrimStatus.txt as a proxy) and divide it by the original trimmed read length, there is no bias either...
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Old 05-18-2010, 06:00 AM   #3
lzembek
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Thanks for checking! I'm curious, how are you shearing after circularization? Covaris or nebulization? Also, are you using carrier DNA? It shouldn't make a difference, I just want to rule anything put...
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Old 05-24-2010, 11:31 PM   #4
grace033
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In solexa sequencing, pair end library especially index PE, we also found that pollution of read1 adpter >>read2, i don‘t understand why?
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Old 05-24-2010, 11:34 PM   #5
grace033
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BTW, index mate pair library !
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Old 06-03-2010, 03:32 AM   #6
flxlex
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Quote:
Originally Posted by lzembek View Post
Thanks for checking! I'm curious, how are you shearing after circularization? Covaris or nebulization? Also, are you using carrier DNA? It shouldn't make a difference, I just want to rule anything put...
We follow the standard protocols, so nebulization and carrier DNA.
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