MS-qFRET: A quantum dot-based method for analysis of DNA methylation

http://genome.cshlp.org/content/early/2009/05/14/gr.088831.108
Vasudev J Bailey1, Hariharan Easwaran1, Yi Zhang1, Elizabeth Griffiths1,
Steven A Belinsky2, James G. Herman1, Stephen Baylin1, Hetty Carraway1 and
Jeff Wang1,3
E-mail: thwang@jhu.edu

Abstract

DNA methylation contributes to carcinogenesis by silencing key tumor suppressor genes. Here we report an ultrasensitive and reliable nanotechnology assay, MS-qFRET, for detection and quantification of DNA methylation. Bisulfite modified DNA is subjected to PCR amplification with primers that would differentiate between methylated and unmethylated DNA. Quantum dots are then used to capture PCR amplicons and determine the methylation status via fluorescence resonance energy transfer (FRET). Key features of MS-qFRET include its low intrinsic background noise, high resolution and high sensitivity. This approach detects as little as 15 pg of methylated DNA in the presence of a 10,000-fold excess of unmethylated alleles, enables reduced use of PCR (8 cycles), and allows for multiplexed analyses. The high sensitivity of MS-qFRET enables one-step detection of methylation at PYCARD, CDKN2B and CDKN2A genes in patient sputum samples that contain low concentrations of methylated DNA, which normally would require a nested PCR approach. The direct application of MS-qFRET on clinical samples offers great promise for its translational use in early cancer diagnosis, prognostic assessment of tumor behavior, as well as monitoring response to therapeutic agents.

Footnotes

• • Received November 3, 2008.
  • Accepted April 28, 2009.
• Copyright © 2009, Cold Spring Harbor Laboratory Press

Shifted Transversal Design smart-pooling for high coverage interactome mapping

http://genome.cshlp.org/content/early/2009/05/15/gr.090019.108

Xiaofeng Xin1,4, Jean-François Rual2,5, Tomoko Hirozane-Kishikawa2, David E. ill2,
Marc Vidal2,6, Charles Boone1,6 and Nicolas Thierry-Mieg3,4,6
E-mail Nicolas.Thierry-Mieg@imag.fr;
E-mail marc_vidal@dfci.harvard.edu;

Abstract

“Smart-pooling,” in which test reagents are multiplexed in a highly redundant manner, is a promising strategy for achieving high efficiency, sensitivity, and specificity in systems-level projects. However, previous applications relied on low redundancy designs that do not leverage the full potential of smart-pooling, and more powerful theoretical constructions, such as the Shifted Transversal Design (STD), lack experimental validation. Here we evaluate STD smart-pooling in yeast two-hybrid (Y2H) interactome mapping. We employed two STD designs and two established methods to perform ORFeome-wide Y2H screens with 12 baits. We found that STD pooling achieves similar levels of sensitivity and specificity as one-on-one array-based Y2H, while the costs and workloads are divided by three. The screening-sequencing approach is the most cost- and labor-efficient, yet STD identifies about twofold more interactions. Screening-sequencing remains an appropriate method for quickly producing low-coverage interactomes, while STD pooling appears as the method of choice for obtaining maps with higher coverage.

• Supplemental material is available online at www.genome.org. The protein interactions from this publication have been submitted to the IMEx (http://imex.sf.net) Consortium through IntAct (PMID 17145710) and assigned the identifier IM-11695.]

• Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.090019.108.

• • Received December 12, 2008.
  • Accepted April 14, 2009.
• Copyright © 2009 by Cold Spring Harbor Laboratory Press

Combinatorial algorithms for structural variation detection in high-throughput sequenced genomes

http://genome.cshlp.org/content/early/2009/05/15/gr.088633.108
Fereydoun Hormozdiari1,4, Can Alkan2,3,4, Evan E. Eichler2,3,5 and S. Cenk Sahinalp1,5
E-mail eee@gs.washington.edu

Abstract

Recent studies show that along with single nucleotide polymorphisms and small indels, larger structural variants among human individuals are common. The Human Genome Structural Variation Project aims to identify and classify deletions, insertions, and inversions (>5 Kbp) in a small number of normal individuals with a fosmid-based paired-end sequencing approach using traditional sequencing technologies. The realization of new ultra-high-throughput sequencing platforms now makes it feasible to detect the full spectrum of genomic variation among many individual genomes, including cancer patients and others suffering from diseases of genomic origin. Unfortunately, existing algorithms for identifying structural variation (SV) among individuals have not been designed to handle the short read lengths and the errors implied by the “next-gen” sequencing (NGS) technologies. In this paper, we give combinatorial formulations for the SV detection between a reference genome sequence and a next-gen-based, paired-end, whole genome shotgun-sequenced individual. We describe efficient algorithms for each of the formulations we give, which all turn out to be fast and quite reliable; they are also applicable to all next-gen sequencing methods (Illumina, 454 Life Sciences [Roche], ABI SOLiD, etc.) and traditional capillary sequencing technology. We apply our algorithms to identify SV among individual genomes very recently sequenced by Illumina technology.

• [Supplemental material is available online at www.genome.org. The source code of the algorithm implementations and predicted structural variants are available at http://compbio.cs.sfu.ca/strvar.htm.]

• Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.088633.108.

• • Received October 26, 2008.
  • Accepted April 10, 2009.
• Copyright © 2009 by Cold Spring Harbor Laboratory Press

Overlapping pools for high-throughput targeted resequencing

http://genome.cshlp.org/content/early/2009/05/15/gr.088559.108
Snehit Prabhu,1 and Itsik Pe'er,1
E-mail snehitp@columbia.edu

Abstract

Resequencing genomic DNA from pools of individuals is an effective strategy to detect new variants in targeted regions and compare them between cases and controls. There are numerous ways to assign individuals to the pools on which they are to be sequenced. The naïve, disjoint pooling scheme (many individuals to one pool) in predominant use today offers insight into allele frequencies, but does not offer the identity of an allele carrier. We present a framework for overlapping pool design, where each individual sample is resequenced in several pools (many individuals to many pools). Upon discovering a variant, the set of pools where this variant is observed reveals the identity of its carrier. We formalize the mathematical framework for such pool designs and list the requirements from such designs. We specifically address three practical concerns for pooled resequencing designs: (1) false-positives due to errors introduced during amplification and sequencing; (2) false-negatives due to undersampling particular alleles aggravated by nonuniform coverage; and consequently, (3) ambiguous identification of individual carriers in the presence of errors. We build on theory of error-correcting codes to design pools that overcome these pitfalls. We show that in practical parameters of resequencing studies, our designs guarantee high probability of unambiguous singleton carrier identification while maintaining the features of naïve pools in terms of sensitivity, specificity, and the ability to estimate allele frequencies. We demonstrate the ability of our designs in extracting rare variations using short read data from the 1000 Genomes Pilot 3 project.

• • Received October 25, 2008.
  • Accepted March 27, 2009.
• Copyright © 2009 by Cold Spring Harbor Laboratory Press

Domain shuffling and the evolution of vertebrates

http://genome.cshlp.org/content/early/2009/05/14/gr.087072.108
Takeshi Kawashima1, Shuichi Kawashima2, Chisaki Tanaka3, Miho Murai4,
Masahiko Yoneda4, Nicholas H Putnum5, Daniel S Rokhsar5, Minoru Kanehisa6,
Nori Satoh7 and Hiroshi Wada3,8
E-mail: 98champ@msg.biglobe.ne.jp

Abstract

The evolution of vertebrates was driven by a number of important events, including the development of cartilage, the immune system, and complicated craniofacial structures. Here we examine domain shuffling as an alternative mechanism for the contribution of novel genetic material required for vertebrate evolution. We mapped domain-shuffling events during the evolution of deuterostomes with a focus on how domain shuffling contributed to the evolution of vertebrate- and chordate-specific characteristics. We identified approximately 1,000 new domain pairs in the vertebrate lineage, including approximately 100 that were shared by all seven of the vertebrate species examined. Some of these pairs occur in the protein components of vertebrate-specific structures, such as cartilage and the inner ear, suggesting that domain shuffling made a marked contribution to the evolution of vertebrate-specific characteristics. The evolutionary history of the domain pairs is traceable; for example, the Xlink domain of aggrecan, one of the major components of cartilage, was originally utilized as a functional domain of a surface molecule of blood cells in protochordate ancestors, and it was recruited by the protein of the matrix component of cartilage in the vertebrate ancestor. We also identified genes that were created as a result of domain shuffling in ancestral chordates. Some of these are involved in the functions of chordate structures, such as the endostyle, Reissner's fiber of the neural tube, and the notochord. Our analyses shed new light on the evolutionary role of domain shuffling, especially in the evolution of vertebrates and chordates.

Footnotes

• • Received September 23, 2008.
  • Accepted April 24, 2009.
• Copyright © 2009, Cold Spring Harbor Laboratory Press

DNA Sudoku—harnessing high-throughput sequencing for multiplexed specimen analysis

http://genome.cshlp.org/content/early/2009/05/15/gr.092957.109
Yaniv Erlich, Kenneth Chang, Assaf Gordon, Roy Ronen, Oron Navon, Michelle Rooks and Gregory J. Hannon,1
E-mail hannon@cshl.edu;

Abstract

Next-generation sequencers have sufficient power to analyze simultaneously DNAs from many different specimens, a practice known as multiplexing. Such schemes rely on the ability to associate each sequence read with the specimen from which it was derived. The current practice of appending molecular barcodes prior to pooling is practical for parallel analysis of up to many dozen samples. Here, we report a strategy that permits simultaneous analysis of tens of thousands of specimens. Our approach relies on the use of combinatorial pooling strategies in which pools rather than individual specimens are assigned barcodes. Thus, the identity of each specimen is encoded within the pooling pattern rather than by its association with a particular sequence tag. Decoding the pattern allows the sequence of an original specimen to be inferred with high confidence. We verified the ability of our encoding and decoding strategies to accurately report the sequence of individual samples within a large number of mixed specimens in two ways. First, we simulated data both from a clone library and from a human population in which a sequence variant associated with cystic fibrosis was present. Second, we actually pooled, sequenced, and decoded identities within two sets of 40,000 bacterial clones comprising approximately 20,000 different artificial microRNAs targeting Arabidopsis or human genes. We achieved greater than 97% accuracy in these trials. The strategies reported here can be applied to a wide variety of biological problems, including the determination of genotypic variation within large populations of individuals.