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Old 05-04-2010, 02:00 AM   #1
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Default PubMed: Titration-free massively parallel pyrosequencing using trace amounts of start

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Titration-free massively parallel pyrosequencing using trace amounts of starting material.

Nucleic Acids Res. 2010 Apr 30;

Authors: Zheng Z, Advani A, Melefors O, Glavas S, Nordström H, Ye W, Engstrand L, Andersson AF

Continuous efforts have been made to improve next-generation sequencing methods for increased robustness and for applications on low amounts of starting material. We applied double-stranded library protocols for the Roche 454 platform to avoid the yield-reducing steps associated with single-stranded library preparation, and applied a highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) method. The MGB-probe qPCR, which can detect as low as 100 copies, was used to quantify the amount of effective library, i.e. molecules that form functional clones in emulsion PCR. We also demonstrate that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labour-intensive and costly titration can be eliminated and trace amounts of starting material such as precious clinical samples, transcriptomes of small tissue samples and metagenomics on low biomass environments is applicable.

PMID: 20435675 [PubMed - as supplied by publisher]



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Old 05-06-2010, 10:24 AM   #2
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We have been considering using KAPA qPCR for quatitating our lib. Has anyone done some comparisons among different qPCRs? Anyone uses KAPA kit and how much copy-per-bead is optimal?
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Old 05-06-2010, 12:26 PM   #3
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You might want to repost your question in the Sample Prep forum!
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Old 05-06-2010, 12:41 PM   #4
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Good idea, thanks!
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