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{userid:"frank.troxell", "refid":"110","repocollections":"","attachment":"","_thumb":"","articletype":"article","sectionheading":"","title":"Multiplex Picoliter-Droplet Digital PCR for Quantitative Assessment of DNA Integrity in Clinical Samples","year":"2013","author":"A Didelot, S K Kotsopoulos, A Lupo, D Pekin, X Li, I Atochin, P Srinivasan, Q Zhong, J Olson, D R Link, P Laurent-Puig, H Blons, J B Hutchison, V Taly","journal":"Clin Chem","volume":"","number":"","pages":"","month":"February","doi":"10.1373\/clinchem.2012.193409","pubmed":"23403697","pdflink":"","urllink":"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/23403697","abstract":"BACKGROUND: Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA.METHODS: Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3 kb. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues.RESULTS: One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and <\/=1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants.CONCLUSIONS: The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.","note":"Didelot, Audrey xD;Kotsopoulos, Steve K xD;Lupo, Audrey xD;Pekin, Deniz xD;Li, Xinyu xD;Atochin, Ivan xD;Srinivasan, Preethi xD;Zhong, Qun xD;Olson, Jeff xD;Link, Darren R xD;Laurent-Puig, Pierre xD;Blons, Helene xD;Hutchison, J Brian xD;Taly, Valerie xD;ENG xD;2013\/02\/14 06:00 xD;Clin Chem. 2013 Feb 12.","tags":"System, Ion AmpliSeq DNA Panel,\tIon PGM,  Ion 318 Chip,  AmpliSeq Cancer HotSpot Panel, Basic Research, PGM, Genome, Next generation sequencing, sequencing, System, Ion AmpliSeq DNA Panel,Ion PGM,Ion 318 Chip, AmpliSeq Cancer HotSpot Panel,Basic Research","publisher":"","booktitle":"","editor":"","address":"","school":"","issn":"","isi":"","key":"Didelot2013","howpublished":"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/23403697"}
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