Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting

Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery.

[1]  J. Harrow,et al.  A conditional knockout resource for the genome-wide study of mouse gene function , 2011, Nature.

[2]  A. Stewart,et al.  Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis. , 2011, Methods.

[3]  E. Lander.  Initial impact of the sequencing of the human genome , 2011, Nature.

[4]  Andrew C. Adey,et al.  Haplotype-resolved genome sequencing of a Gujarati Indian individual , 2011, Nature Biotechnology.

[5]  Stephen R Quake,et al.  Whole-genome molecular haplotyping of single cells , 2011, Nature Biotechnology.

[6]  V. Bansal,et al.  The next phase in human genetics , 2011, Nature Biotechnology.

[7]  Kenny Q. Ye,et al.  Mapping copy number variation by population scale genome sequencing , 2010, Nature.

[8]  P. D. de Jong,et al.  Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination , 2010, Journal of biomedicine & biotechnology.

[9]  Douglas A. Bell,et al.  Human single-nucleotide polymorphisms alter p53 sequence-specific binding at gene regulatory elements , 2010, Nucleic Acids Res..

[10]  Edwin Cuppen,et al.  Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples , 2010, Nature Methods.

[11]  Peter H. Sudmant,et al.  Diversity of Human Copy Number Variation and Multicopy Genes , 2010, Science.

[12]  A. Stewart,et al.  Single-stranded heteroduplex intermediates in λ Red homologous recombination , 2010, BMC Molecular Biology.

[13]  Zhongming Zhao,et al.  Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer , 2010, Nucleic acids research.

[14]  Evan E. Eichler,et al.  LINE-1 Retrotransposition Activity in Human Genomes , 2010, Cell.

[15]  Andrew F. Neuwald,et al.  Natural Mutagenesis of Human Genomes by Endogenous Retrotransposons , 2010, Cell.

[16]  Hsu-hsin Chen,et al.  A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells. , 2010, Cell stem cell.

[17]  Emily H Turner,et al.  Target-enrichment strategies for next-generation sequencing , 2010, Nature Methods.

[18]  Benjamin P. Blackburne,et al.  Mutation spectrum revealed by breakpoint sequencing of human germline CNVs , 2010, Nature Genetics.

[19]  M. Gerstein,et al.  Variation in Transcription Factor Binding Among Humans , 2010, Science.

[20]  P. Andrews,et al.  Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system , 2010, In Vitro Cellular & Developmental Biology - Animal.

[21]  K. Frazer,et al.  Microdroplet-based PCR amplification for large scale targeted sequencing , 2009, Nature Biotechnology.

[22]  Yang Xu,et al.  Modeling disease in human ESCs using an efficient BAC-based homologous recombination system. , 2010, Cell stem cell.

[23]  A. F. Stewart,et al.  A recombineering pipeline to make conditional targeting constructs. , 2010, Methods in enzymology.

[24]  Yong-shu He,et al.  [Structural variation in the human genome]. , 2009, Yi chuan = Hereditas.

[25]  Paul Medvedev,et al.  Computational methods for discovering structural variation with next-generation sequencing , 2009, Nature Methods.

[26]  A. Visel,et al.  Genomic Views of Distant-Acting Enhancers , 2009, Nature.

[27]  Dmitry Pushkarev,et al.  Single-molecule sequencing of an individual human genome , 2009, Nature Biotechnology.

[28]  K. Frazer,et al.  Human genetic variation and its contribution to complex traits , 2009, Nature Reviews Genetics.

[29]  L. Liang,et al.  Mapping complex disease traits with global gene expression , 2009, Nature Reviews Genetics.

[30]  Joshua M. Korn,et al.  Mapping and sequencing of structural variation from eight human genomes , 2008, Nature.

[31]  Karl Mechtler,et al.  BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals , 2008, Nature Methods.

[32]  J. Lupski,et al.  The complete genome of an individual by massively parallel DNA sequencing , 2008, Nature.

[33]  W. Donahue,et al.  Fosmid Libraries for Genomic Structural Variation Detection , 2007, Current protocols in human genetics.

[34]  D. Altshuler,et al.  Completing the map of human genetic variation , 2007, Nature.

[35]  E. Eichler,et al.  Population Stratification of a Common APOBEC Gene Deletion Polymorphism , 2007, PLoS genetics.

[36]  A. Hyman,et al.  A recombineering pipeline for functional genomics applied to Caenorhabditis elegans , 2006, Nature Methods.

[37]  Mihail Sarov,et al.  An improved recombineering approach by adding RecA to lambda Red recombination. , 2006, Molecular biotechnology.

[38]  Mihail Sarov,et al.  An improved recombineering approach by adding RecA to λ Red recombination , 2006 .

[39]  S. Cardona,et al.  An expression vector containing a rhamnose-inducible promoter provides tightly regulated gene expression in Burkholderia cenocepacia. , 2005, Plasmid.

[40]  E. Eichler,et al.  Fine-scale structural variation of the human genome , 2005, Nature Genetics.

[41]  P. Holland,et al.  Eleven daughters of NANOG. , 2004, Genomics.

[42]  B. Seed,et al.  Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes , 2003, Nature Biotechnology.

[43]  V. Beneš,et al.  Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles , 2003, Nature Biotechnology.

[44]  A. Stewart,et al.  Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells , 2003, BMC Molecular Biology.

[45]  W. Szybalski,et al.  Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. , 2002, Genome research.

[46]  S. Elledge,et al.  Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step , 2002, Nature Genetics.

[47]  H. Matthes,et al.  Murine inter-strain polymorphisms alter gene targeting frequencies at the mu opioid receptor locus in embryonic stem cells , 2001, Mammalian Genome.

[48]  D. Court,et al.  High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[49]  D. Court,et al.  A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. , 2001, Genomics.

[50]  Giuseppe Testa,et al.  DNA cloning by homologous recombination in Escherichia coli , 2000, Nature Biotechnology.

[51]  V. Beneš,et al.  Point mutation of bacterial artificial chromosomes by ET recombination , 2000, EMBO reports.

[52]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[53]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[54]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.