Massively parallel haplotyping on microscopic beads for the high-throughput phase analysis of single molecules.

PloS One
Jérôme BoulangerIrene Tiemann-Boege

Abstract

In spite of the many advances in haplotyping methods, it is still very difficult to characterize rare haplotypes in tissues and different environmental samples or to accurately assess the haplotype diversity in large mixtures. This would require a haplotyping method capable of analyzing the phase of single molecules with an unprecedented throughput. Here we describe such a haplotyping method capable of analyzing in parallel hundreds of thousands single molecules in one experiment. In this method, multiple PCR reactions amplify different polymorphic regions of a single DNA molecule on a magnetic bead compartmentalized in an emulsion drop. The allelic states of the amplified polymorphisms are identified with fluorescently labeled probes that are then decoded from images taken of the arrayed beads by a microscope. This method can evaluate the phase of up to 3 polymorphisms separated by up to 5 kilobases in hundreds of thousands single molecules. We tested the sensitivity of the method by measuring the number of mutant haplotypes synthesized by four different commercially available enzymes: Phusion, Platinum Taq, Titanium Taq, and Phire. The digital nature of the method makes it highly sensitive to detecting haplotype ratios of les...Continue Reading

References

Apr 11, 1990·Nucleic Acids Research·A MeyerhansS Wain-Hobson
Jun 11, 1995·Nucleic Acids Research·S J OdelbergR White
May 30, 1998·Nucleic Acids Research·M S JudoC Wilson
Jan 29, 1999·Journal of Virological Methods·G FangB Weiser
Feb 13, 2001·Clinica Chimica Acta; International Journal of Clinical Chemistry·F V ShammasA Osland
Mar 22, 2002·Nature Reviews. Cancer·A G Knudson
May 6, 2003·Proceedings of the National Academy of Sciences of the United States of America·Robi D MitraGeorge M Church
Jun 13, 2003·Proceedings of the National Academy of Sciences of the United States of America·Chunming Ding, Charles R Cantor
Jul 15, 2003·Proceedings of the National Academy of Sciences of the United States of America·Devin DressmanBert Vogelstein
Aug 9, 2003·Science·Jun ZhuGeorge M Church
Dec 3, 2004·Applied and Environmental Microbiology·Shinya KurataYoichi Kamagata
Aug 6, 2005·Science·Jay ShendureGeorge M Church
Oct 20, 2005·Journal of Bioscience and Bioengineering·Takahiro Kanagawa
Oct 29, 2005·Nature·UNKNOWN International HapMap Consortium
Nov 1, 2005·Proceedings of the National Academy of Sciences of the United States of America·Frank DiehlBert Vogelstein
Jan 25, 2006·Nature Methods·Meng LiKenneth W Kinzler
Jun 23, 2006·Nature Methods·Richard WilliamsAndrew D Griffiths
Feb 20, 2008·BMC Genomics·Matthew J HaydenKenneth J Chalmers
Jun 20, 2008·Nucleic Acids Research·Daniel J TurnerMatthew E Hurles
Aug 5, 2008·Genome Research·Vikas BansalVineet Bafna
Feb 10, 2009·Nature Methods·Ming XiaoPui-Yan Kwok
Apr 22, 2009·International Journal of Biological Sciences·Zhihua JiangRichard M Harland
Sep 1, 2009·Annual Review of Genomics and Human Genetics·Yun LiGonçalo Abecasis
Dec 18, 2009·Nature·Augustine KongKari Stefansson
Mar 3, 2010·Analytical Chemistry·Yong ZengRichard A Mathies
Oct 29, 2010·Nature·Gonçalo R AbecasisGil A McVean
Dec 21, 2010·Nature Biotechnology·Jacob O KitzmanJay Shendure
Dec 21, 2010·Nature Biotechnology·H Christina FanStephen R Quake
Feb 9, 2011·Nature Reviews. Genetics·Ryan TewheyNicholas J Schork
Oct 19, 2011·Journal of Bioprocessing & Biotechniques·Hirendra Nath BanerjeeZack Hawkins

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Citations

Jun 7, 2013·Human Molecular Genetics·Deepali N ShindeIrene Tiemann-Boege
Feb 18, 2016·Scientific Reports·Thomas W LaverMichael N Weedon
Mar 15, 2015·BioMed Research International·Barbara ArbeithuberIrene Tiemann-Boege
Aug 2, 2016·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·Barbara ArbeithuberIrene Tiemann-Boege
Feb 13, 2021·NAR Genomics and Bioinformatics·Gundula PovysilIrene Tiemann-Boege

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Methods Mentioned

BETA
PCR
454 sequencing
Genotyping
Assay

Software Mentioned

Mathworks
BEH
Metamorph
Matlab

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