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Old 02-26-2010, 11:15 AM   #1
kmcarr
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Default Extreme nucleotide bias at fragment ends of Illumina mate pair library

We recently prepped and ran our first (and thus far only) Illumina mate pair run. There were seven libraries prepped, all different isolates of E. coli genomic DNA. The Illumina mate pair kit was used and standard protocol followed. Both the initial (3 kbp) and second fragmentation were performed using a nebulizer. According to the lab personnel nothing unusual was noted during prep.

After sequencing an extreme bias for T at the 5' ends of both reads was observed (see the attached % Base Calls plot). These are NOT errors in sequencing, the full read align with high fidelity to the E. coli genome. The reads align in a fairly uniform manner around the whole genome but obviously are not random with respect to the positioning of the 5' end of the reads. Mapping shows the vast majority of sequenced fragments to be properly constructed mate pairs; they are separated by ~2,700 - 3,000 bp and point away from each other. All seven of the libraries look like this.

We have a hypothesis about cause but I'd like to ask the community if they have ever seen anything like this with an Illumina mate pair library.

Here is our hypothesis: After the large fragments are circularized there is an exonuclease step to degrade and remaining, linear DNA. The exonuclease is supplied with the mate pair prep kit so I don't know specifically which exo it is. After this digestion the sample is heated @70C for 30' to kill the exo, followed by addition of EDTA to 20mM. This if followed by nebulization to fragment the circles, followed by streptavidin selection of the junction fragment and sequencing adapter ligation. We suspect that there was some very small amount of residual exo activity left after heat inactivation. The random ends created by the subsequent nebulization were acted upon by the exo, with its activity much lower when it encounters a thymidine (or di-thymidine) at the 5' ends of dsDNA. Does this sound reasonable (or at least plausible)? Can any of you good folks think of an alternative mechanism?

Thanks.
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File Type: pdf MatePairFragmentEndBias.pdf (28.0 KB, 106 views)
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Old 03-17-2011, 12:07 PM   #2
dsbreak
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Might it be a bias in where the biotinylation occurs? If the Biotin dNTP mix truly only contains Biotin-dATP[1], that would explain the T bias.

[1] http://seqanswers.com/forums/archive...hp/t-4608.html
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Old 03-17-2011, 12:43 PM   #3
kmcarr
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Quote:
Originally Posted by dsbreak View Post
Might it be a bias in where the biotinylation occurs? If the Biotin dNTP mix truly only contains Biotin-dATP[1], that would explain the T bias.

[1] http://seqanswers.com/forums/archive...hp/t-4608.html
The biotin is incorporated at the ends of the large fragments prior to circularization. The T bias was seen at the ends of the small fragments created after circularization and refragmentation. At this point the biotinylated dA's would be in the middle of the fragments.

As it happens we just completed a mate-pair run, our first since I posted about this problem a year ago. This time there was no evidence of nucleotide bias at the ends of our reads. Never did get a definitive answer as to the cause of the problem from Illumina. As far as I'm concerned my original hypothesis above is still the best bet.
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Old 03-17-2011, 02:03 PM   #4
dsbreak
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I see, I misunderstood. Does this argue against hypothesis? pmid 6752885
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