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Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid tegument protein encoded by ORF51 of the EHV-1 genome. EHV-1 UL11 was previously reported by other researchers using the RacL22 and RacH strains to be nonessential for viral replication in cultured cells. Here, we constructed UL11 mutant viruses including a UL11 null mutant and three C-terminal truncated mutants, for further characterization of EHV-1 UL11 using bacterial artificial chromosome (BAC) technology based on the neuropathogenic strain Ab4p. EHV-1 Ab4p UL11 was localized to juxtanuclear and Golgi regions as reported by other researchers. We found that no progeny viruses were produced by transfection of fetal equine kidney cells and rabbit kidney (RK-13) cells with the UL11 null mutant and truncation mutant BAC DNAs. However, mutant viruses were generated after transfection of RK13-UL11 cells constitutively expressing EHV-1 UL11 with the mutant BAC DNAs. In conclusion, UL11 of EHV-1 Ab4p is essential for replication in cultured cells.

Prenatal diagnosis focuses on the detection of anatomic and physiologic problems with a foetus before birth. Karyotyping is currently considered the gold standard for prenatal diagnosis of chromosomal abnormalities, but this method can be time consuming. This study evaluated the diagnostic accuracy of the BACs-on-Beads TM (BoBs™) assay for the rapid diagnosis of aneuploidies and microdeletions. A total of 625 samples from pregnant women in Fujian province, in southeastern China-including three chorionic villus biopsies, 523 amniotic fluid samples, and 99 umbilical-cord centesis samples-were assessed for chromosomal abnormalities by karyotyping and by the BoBs™ assay. A diagnosis was successfully achieved by karyotyping for 98.8% (618/625) and by the BoBs™ assay for 100% (625/625) of the samples. Both assays were concordant for trisomy 21 (2.72%, 17/625), trisomy 18 (1.12%, 7/625), trisomy 13 (0.48%, 3/625), and sex chromosome aneuploidies (0.8%, 5/625). Unlike karyotyping, the BoBs™ assay detected 22q11.2 microdeletion (0.64%, 4/625), 22q11.2 microduplication (0.16%, 1/625), Smith-Magenis syndrome microdeletion (0.16%, 1/625), and Miller-Dieker syndrome microdeletion (0.16%, 1/625). Thus, the BoBs™ assay is a reliable and rapid test for detecting common aneuploidies and microdeletions for prenatal diagnosis, and could be used instead of karyotyping for detection of common aneuploidies as well as to provide additional information regarding microdeletions., (© 2017 Wiley Periodicals, Inc.)

Background: Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts., Methods: The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids., Results: We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%., Conclusions: AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist's toolbox.

The genome of the cattle tick Rhipicephalus microplus, an ectoparasite with global distribution, is estimated to be 7.1Gbp in length and consists of approximately 70% repetitive DNA. We report the draft assembly of a tick genome that utilized a hybrid sequencing and assembly approach to capture the repetitive fractions of the genome. Our hybrid approach produced an assembly consisting of 2.0Gbp represented in 195,170 scaffolds with a N50 of 60,284bp. The Rmi v2.0 assembly is 51.46% repetitive with a large fraction of unclassified repeats, short interspersed elements, long interspersed elements and long terminal repeats. We identified 38,827 putative R. microplus gene loci, of which 24,758 were protein coding genes (≥100 amino acids). OrthoMCL comparative analysis against 11 selected species including insects and vertebrates identified 10,835 and 3,423 protein coding gene loci that are unique to R. microplus or common to both R. microplus and Ixodes scapularis ticks, respectively. We identified 191 microRNA loci, of which 168 have similarity to known miRNAs and 23 represent novel miRNA families. We identified the genomic loci of several highly divergent R. microplus esterases with sequence similarity to acetylcholinesterase. Additionally we report the finding of a novel cytochrome P450 CYP41 homolog that shows similar protein folding structures to known CYP41 proteins known to be involved in acaricide resistance., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Histone deacetylases (HDACs) play crucial roles during mammalian development and for cellular homeostasis. In addition, these enzymes are promising targets for small molecule inhibitors in the treatment of cancer and neurological diseases. Conditional HDAC knock-out mice are excellent tools for defining the functions of individual HDACs in vivo and for identifying the molecular targets of HDAC inhibitors in disease. Here, we describe the generation of tissue-specific HDAC knock-out mice and delineate a strategy for the generation of conditional HDAC knock-in mice.

The localization of a protein is intrinsically linked to its role in the structural and functional organization of the cell. Advances in transgenic technology have streamlined the use of protein localization as a function discovery tool. Here we review the use of large genomic DNA constructs such as bacterial artificial chromosomes as a transgenic platform for systematic tag-based protein function exploration., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics., (© The Author 2016. Published by Oxford University Press.)

We have developed a new, sequence-specific DNA labeling strategy that will dramatically improve DNA mapping in complex and structurally variant genomic regions, as well as facilitate high-throughput automated whole-genome mapping. The method uses the Cas9 D10A protein, which contains a nuclease disabling mutation in one of the two nuclease domains of Cas9, to create a guide RNA-directed DNA nick in the context of an in vitro-assembled CRISPR-CAS9-DNA complex. Fluorescent nucleotides are then incorporated adjacent to the nicking site with a DNA polymerase to label the guide RNA-determined target sequences. This labeling strategy is very powerful in targeting repetitive sequences as well as in barcoding genomic regions and structural variants not amenable to current labeling methods that rely on uneven distributions of restriction site motifs in the DNA. Importantly, it renders the labeled double-stranded DNA available in long intact stretches for high-throughput analysis in nanochannel arrays as well as for lower throughput targeted analysis of labeled DNA regions using alternative methods for stretching and imaging the labeled long DNA molecules. Thus, this method will dramatically improve both automated high-throughput genome-wide mapping as well as targeted analyses of complex regions containing repetitive and structurally variant DNA., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The National Institute of Genetics Mouse Genome database (NIG_MoG; http://molossinus.lab.nig.ac.jp/msmdb/) primarily comprises the whole-genome sequence data of two inbred mouse strains, MSM/Ms and JF1/Ms. These strains were established at NIG and originated from the Japanese subspecies Mus musculus molossinus. NIG_MoG provides visualized genome polymorphism information, browsing single-nucleotide polymorphisms and short insertions and deletions in the genomes of MSM/Ms and JF1/Ms with respect to C57BL/6J (whose genome is predominantly derived from the West European subspecies M. m. domesticus). This allows users, especially wet-lab biologists, to intuitively recognize intersubspecific genome divergence in these mouse strains using visual data. The database also supports the in silico screening of bacterial artificial chromosome (BAC) clones that contain genomic DNA from MSM/Ms and the standard classical laboratory strain C57BL/6N. NIG_MoG is thus a valuable navigator for exploring mouse genome polymorphisms and BAC clones that are useful for studies of gene function and regulation based on intersubspecific genome divergence.

The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.

BAC clones containing the entire genomic region of a gene including the long-range regulatory elements are very useful for gene functional analysis. However, large genes often span more than the insert of a BAC clone, and single BACs covering the entire region of interest are not available. Here, we describe a general system for linking two or more overlapping BACs into a single clone. Two rounds of homologous recombination are used. In the first, the BAC inserts are subcloned into the pBACLink vectors. In the second, the two BACs are combined together. Multiple BACs in a contig can be combined by alternating use of the pBACLInk vectors, resulting in several BAC clones containing as much of the genomic region of a gene as required. Such BACs can then be used in gene expression studies and/or gene therapy applications.

Copy number variations (CNVs) are structural variations of the human genome. These alterations result in variant copy numbers of certain stretches of DNA. In other words, some regions may be present in more or less copies than in a reference genome; however, these copy number changes do not have any impact on the phenotype. Also, CNVs may be extremely large and cytogenetically detectable or submicroscopic but still spanning several megabasepairs (Mb). In the recent years, array technology has identified especially the latter ones as so-called copy number variant (CNV) polymorphisms. These CNVs are detected in ~12 % of the human genome sequences and may comprise several hundred kilobasepairs. CNVs contribute significantly to the inter-individual differences in humans, and can range between 0.5 and 1.5 Mb amongst different genomes, well within the level of detection using cytogenetics techniques. Thus, they can be visualized by FISH using bacterial artificial chromosomes (BACs) as probes. Here we describe a method that enables discrimination of individual homologous chromosomes at the single cell level based on CNVs in the genome, called parental origin determination fluorescence in situ hybridization (POD-FISH). Possible fields of applications of this single cell-directed approach are in analyses of the parental origin of single chromosomes in inherited and acquired chromosomal aberrations.

Many of the experimental approaches aimed at studying gene function heavily rely on the ability to make precise modifications in the gene's DNA sequence. Homologous recombination (HR)-based strategies provide a convenient way to create such types of modifications. HR-based DNA sequence manipulations can be enormously facilitated by expressing in E. coli a small set of bacteriophage proteins that make the exchange of DNA between a linear donor and the target DNA molecules extremely efficient. These in vivo recombineering techniques have been incorporated as essential components of the molecular toolbox in many model organisms. In this chapter, we describe the experimental procedures involved in recombineering-based tagging of an Arabidopsis gene contained in a plant transformation-ready bacterial artificial chromosome (TAC).

Glioblastoma multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSVs) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered four copies of the recognition sequence for miR-124 into the 3'UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.

The isolation of germ line competent mouse Embryonic Stem (ES) cells and the ability to modify the genome by homologous recombination has revolutionized life science research. Since its initial discovery, several approaches have been introduced to increase the efficiency of homologous recombination, including the use of isogenic DNA for the generation of targeting constructs, and the use of Bacterial Artificial Chromosomes (BACs). BACs have the advantage of combining long stretches of homologous DNA, thereby increasing targeting efficiencies, with the possibilities delivered by BAC recombineering approaches, which provide the researcher with almost unlimited possibilities to efficiently edit the genome in a controlled fashion. Despite these advantages of BAC targeting approaches, a widespread use has been hampered, mainly because of the difficulties in identifying BAC-targeted knockout alleles by conventional methods like Southern Blotting. Recently, we introduced a novel BAC targeting strategy, in which Restriction Fragment Length Polymorphisms (RFLPs) are targeted in polymorphic mouse ES cells, enabling an efficient and easy PCR-based readout to identify properly targeted alleles. Here we provide a detailed protocol for the generation of targeting constructs, targeting of ES cells, and convenient PCR-based analysis of targeted clones, which enable the user to generate knockout ES cells of almost every gene in the mouse genome within a 2-month period.

A step-by-step detailed procedure is presented to progressively truncate genomic DNA inserts from either end in BACs. The bacterial transposon Tn10 carrying a loxP or a lox511 site is inserted at random into BAC DNA inside the bacterial cell. The cells are then infected with bacteriophage P1. The Cre protein expressed by phage P1 generates end-deletions by specifically recombining the inserted loxP (or lox511) with the loxP (or lox511) endogenous to and flanking insert DNA in BACs from the respective end. The Cre protein also helps phage P1 transduce the BAC DNA by packaging it in P1 heads. This packaging by P1 not only recovers the rare BAC clones containing Tn10 insertions efficiently but also selects end-truncated BACs from those containing inversions of portions of their DNA caused by transposition of Tn10 in the opposite orientation. The libraries of end-deleted BACs generated by this procedure are suitable for numerous mapping studies. Because DNA in front of the loxP (or lox511) arrowheads in the Tn10 transposon is retained at the newly created BAC end, exogenous DNA cassettes such as enhancer-traps and iTol2 ends can be efficiently introduced into BAC ends for germline expression in zebrafish or mice. The methodology should facilitate functional mapping studies of long-range cis-acting gene regulatory sequences in these organisms.

Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.

A critical factor in the study of herpesviruses, their genes and gene functions is the capacity to derive mutants that harbor deletions, truncations, or insertions within the genetic elements of interest. Once constructed the impact of the introduced mutation on the phenotypic properties of the rescued virus can be determined in either in vitro or in vivo systems. However, the construction of such mutants by traditional virological mutagenesis techniques can be a difficult and laborious undertaking. The maintenance of a viral genome as an infectious bacterial artificial chromosome (iBAC), however, endows the capacity to manipulate the viral genome for mutagenesis studies with relative ease. Here, the construction and characterization of two gene deletion mutants of an alphaherpesvirus maintained as iBAC in combination with an inducible homologous recombination system in Escherichia coli is detailed. The methodology is generally applicable to any iBAC and is demonstrated to be a highly efficient and informative approach for mutant virus construction.

Bacterial artificial chromosomes (BACs) can accommodate and stably propagate the genomes of large DNA viruses in E. coli. As DNA virus genomes are often per se infectious upon transfection into mammalian cells, their cloning in BACs and easy modification by homologous recombination in bacteria has become an important strategy to investigate the functions of individual virus genes. This chapter describes a strategy to clone the genomes of viruses of the Alphaherpesvirinae subfamily within the family of the Herpesviridae, which is a group of large DNA viruses that can establish both lytic and latent infections in most animal species including humans. The cloning strategy includes the following steps: (1) Construction of a transfer plasmid that contains the BAC backbone with selection and screening markers, and targeting sequences which support homologous recombination between the transfer plasmid and the alphaherpesvirus genome. (2) Introduction of the transfer plasmid sequences into the alphaherpesvirus genome via homologous recombination in mammalian cells. (3) Isolation of recombinant virus genomes containing the BAC backbone sequences from infected mammalian cells and electroporation into E. coli. (4) Preparation of infectious BAC DNA from bacterial cultures and transfection into mammalian cells. (5) Isolation and characterization of progeny virus.

The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering.

Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

With the advent of BAC recombineering techniques, transcriptional promoter and enhancer isolation studies have become much more feasible in zebrafish than in mouse given the easy access to large numbers of fertilized zebrafish eggs and offspring in general, the easy to follow ex-utero development of zebrafish, an overall less skill demand and a more cost-effective technique. Here we provide guidelines for the generation of BAC recombineering-based transgenic zebrafish for DNA transcriptional promoter and enhancer identification studies as well as protocols for their analysis, which have been successfully applied in our laboratories many times. BAC recombineering in zebrafish allows for economical functional genomics studies, for example by integrating developmental biology with comparative genomics approaches to validate potential enhancer elements of vertebrate transcription factors.

Fluorescence in situ hybridization (FISH) and/or array-comparative genomic hybridization (aCGH) performed after initial banding cytogenetics is still the gold standard for detection of chromosomal rearrangements. Although aCGH provides a higher resolution, FISH has two main advantages over the array-based approaches: (1) it can be applied to characterize balanced as well as unbalanced rearrangements, whereas aCGH is restricted to unbalanced ones, and (2) chromosomal aberrations present in low level or complex mosaics can be characterized by FISH without any problems, while aCGH requires presence of over 50 % of aberrant cells in the sample for detection. Recently, a new FISH-based probe set was presented: the so-called pericentric-ladder-FISH (PCL-FISH) that enables characterization of chromosomal breakpoints especially in mosaic small supernumerary marker chromosomes (sSMC). It can also be applied on large inborn or acquired derivative chromosomes. The main feature of this set is that the probes are applied in a chromosome-specific manner and they align along the chromosome in average intervals of ten megabasepairs. Hence PCL-FISH provides denser coverage and a more precise anchorage on the human DNA-sequence than most other FISH-banding approaches.

FISH (fluorescent in situ hybridization) is a molecular cytogenetic method to detect large-scale genetic alterations in tissue and/or cells. Numerical aberrations (deletions and amplifications) and structural aberrations (translocations and fusions) are detectable. Probes bind complementary to the DNA strand of the region of interest. Subsequently, the probes are detected via fluorochromes and appear as colored dots that can be assessed under the fluorescence microscope.In situ hybridization is divided into three steps: pretreatment, hybridization, and posthybridization. Pretreatment opens up the cell membranes for hybridization, so that the probe can bind to the complementary DNA target. Posthybridization includes washing steps to remove excessive probes and detection of probes via secondary marked fluorochromes. DAPI stains nuclei and serves as mounting media.

Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly.

Genomic instability tends to occur at specific genomic regions known as common fragile sites (FS). FS are evolutionarily conserved and generally involve late replicating regions with AT-rich sequences. The possible correlation between some FS and cancer-related breakpoints emphasizes on the importance of understanding the mechanisms of chromosomal instability at these sites. Although about 230 FS have already been mapped cytogenetically, only a few of them have been characterized on a molecular level. In this chapter, we provide a protocol for mapping of common FS using bacterial artificial chromosome (BAC) probes in fluorescence in situ hybridization (FISH) and suggest the usage of lymphocytes from Fanconi anemia patients as a model system. In the latter, rare FS are expressed much more frequently than in, for example, aphidicolin-induced blood lymphocyte preparations. Knowing the exact location of FS enables the molecular comparison of their location and breakpoints that appear during evolution, cancer development and inherited disorders.

Genome engineering strategies--such as genome editing, reduction and shuffling, and de novo genome synthesis--enable the modification of specific genomic locations in a directed and combinatorial manner. These approaches offer an unprecedented opportunity to study central evolutionary issues in which natural genetic variation is limited or biased, which sheds light on the evolutionary forces driving complex and extremely slowly evolving traits; the selective constraints on genome architecture; and the reconstruction of ancestral states of cellular structures and networks.

The consortium responsible for the sequencing of the tomato (Solanum lycopersicum) genome initially focused on the sequencing of the euchromatic regions using a BAC-by-BAC strategy. We analyzed the compositional features of the whole collection of BAC sequences publically available. This analysis highlights specific peculiarities of heterochromatic and euchromatic BACs, in particular: the whole BAC collection has i) a large variability in repeat and gene content, ii) a positive and significant correlation of LTR retrotransposons of the Gypsy class with the repeat content and iii) the preferential location of the SINEs (short interspersed nuclear elements) in BAC sequences showing a low repeat content. Our results point out a typical design of the tomato chromosomes and pave the way for further investigations on the relationship between DNA primary structure and chromatin organization in Solanaceae genomes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Chromosome conformation capture (3C) is a biochemical assay to reveal higher order chromosomal organizations mediated by physical contact between discrete DNA segments in vivo. Chromosomal organizations are involved in transcriptional regulation of a number of genes in various cell types. We have adapted 3C for analyzing the intrachromosomal looping organization of rod and cone photoreceptor genes in the mammalian retina. Here, we describe a detailed protocol for 3C assays on whole mouse retinas. Using the M-cone opsin gene as an example, we demonstrate how to genetically distinguish 3C signals from cones versus rods in retinal 3C assays. We also describe the challenges and key points of 3C design and performance as well as appropriate controls and result interpretations.

An unresolved bottleneck in bacterial artificial chromosome (BAC) transgenesis is low efficiency generation of founder mice because of suboptimal quality of the manipulated BAC DNA. Using mini-gel electrophoresis and electro-elution that circumvents CsCl(2) centrifugation, column chromatography, and resin purifications, we have used RECOCHIP, a commercially available dialysis cassette for the purification of BAC DNA that generates transgenic founders with up to 80% efficiency.

Background: Bovine herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC), the lack of a complete genome sequence for this strain limits its experimental use., Methods: In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach., Results: The long unique coding region (LUR) consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA). As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G) differs from the other prDNA units (prDNA-inner). Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions., Conclusions: In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other herpesviruses.

Chromosomal distribution of the Fat element that was isolated from bacterial artificial chromosome (BAC) end sequences of wheat chromosome 3B was studied in 45 species representing eight genera of Poaceae (Aegilops, Triticum, Agropyron, Elymus, Secale, Hordeum, Avena and Triticale) using fluorescence in situ hybridisation (FISH). The Fat sequence was not present in oats and in two barley species, Hordeum vulgare and Hordeum spontaneum, that we investigated. Only very low amounts of the Fat element were detected on the chromosomes of two other barley species, Hordeum geniculatum and Hordeum chilense, with different genome compositions. The chromosomes of other cereal species exhibited distinct hybridisation patterns with the Fat probe, and labelling intensity varied significantly depending on the species or genome. The highest amount of hybridisation was detected on chromosomes of the D genome of Aegilops and Triticum and on chromosomes of the S genome of Agropyron. Despite the bioinformatics analysis of several BAC clones that revealed the tandem organisation of the Fat element, hybridisation with the Fat probe produces uneven, diffuse signals in the proximal regions of chromosomes. In some of the genomes we investigated, however, it also forms distinct, sharp clusters in chromosome-specific positions, and the brightest fluorescence was always observed on group 4 chromosomes. Thus, the Fat element represents a new family of Triticeae-specific, highly repeated DNA elements with a clustered-dispersed distribution pattern. These elements may have first emerged in cereal genomes at the time of divergence of the genus Hordeum from the last common ancestor. During subsequent evolution, the amount and chromosomal distribution of the Fat element changed due to amplification, elimination and re-distribution of this sequence. Because the labelling patterns that we detected were highly specific, the Fat element can be used as an accessory probe in FISH analysis for chromosome identification and investigation of evolutionary processes at the chromosomal level.

We synthesized reversible terminators with tethered inhibitors for next-generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.

By applying a method that combines end-sequence profiling and massively parallel sequencing, we obtained a sequence-level map of chromosomal aberrations in the genome of the MCF-7 breast cancer cell line. A total of 157 distinct somatic breakpoints of two distinct types, dispersed and clustered, were identified. A total of 89 breakpoints are evenly dispersed across the genome. A majority of dispersed breakpoints are in regions of low copy repeats (LCRs), indicating a possible role for LCRs in chromosome breakage. The remaining 68 breakpoints form four distinct clusters of closely spaced breakpoints that coincide with the four highly amplified regions in MCF-7 detected by array CGH located in the 1p13.1-p21.1, 3p14.1-p14.2, 17q22-q24.3, and 20q12-q13.33 chromosomal cytobands. The clustered breakpoints are not significantly associated with LCRs. Sequences flanking most (95%) breakpoint junctions are consistent with double-stranded DNA break repair by nonhomologous end-joining or template switching. A total of 79 known or predicted genes are involved in rearrangement events, including 10 fusions of coding exons from different genes and 77 other rearrangements. Four fusions result in novel expressed chimeric mRNA transcripts. One of the four expressed fusion products (RAD51C-ATXN7) and one gene truncation (BRIP1 or BACH1) involve genes coding for members of protein complexes responsible for homology-driven repair of double-stranded DNA breaks. Another one of the four expressed fusion products (ARFGEF2-SULF2) involves SULF2, a regulator of cell growth and angiogenesis. We show that knock-down of SULF2 in cell lines causes tumorigenic phenotypes, including increased proliferation, enhanced survival, and increased anchorage-independent growth.

During mouse meiosis, gene expression and homologous synapsis are intimately linked. Chromosomes that fail to synapse at the zygotene-pachytene transition become transcriptionally silenced by a process called Meiotic Silencing of Unsynapsed Chromatin (MSUC), and this silencing (or defects in it) may in turn cause germ cell losses and infertility. Gene transcription at the chromosomal level can be readily observed using RNA fluorescence in-situ hybridisation (FISH), and this approach allows one to directly compare expression at a specific locus with the synaptic status of the chromosome domain on which it resides. Here we describe a protocol for carrying out RNA FISH on male meiotic cells, together with detail on the important controls and common problems associated with this technique.

Chromosome 14 loss in meningiomas are associated with more aggressive tumour behaviour. To date, no studies have been reported in which the entire chromosome 14q of meningioma tumour cells has been studied by high-resolution array comparative genomic hybridization (a-CGH). Here, we used a high-resolution a-CGH to define the exact localization and extent of numerical changes of chromosome 14 in meningioma patients. An array containing 807 bacterial artificial chromosome clones specific for chromosome 14q (average resolution of approximately 130 Kb) was constructed and applied to the study of 25 meningiomas in parallel to the confirmatory interphase fluorescence in situ hybridization (iFISH) analyses. Overall, abnormalities of chromosome 14q were detected in 10/25 cases (40%). Interestingly, in seven of these cases, loss of chromosome 14q32.3 was detected by iFISH and confirmed to correspond to monosomy 14 by a-CGH. In contrast, discrepant results were found between iFISH and a-CGH in the other three altered cases. In one patient, a diploid background was observed by iFISH, while monosomy 14 was identified by a-CGH. In the remaining two cases, which showed gains of the IGH gene by iFISH, a-CGH did not detected copy number changes in one case showing a tetraploid karyotype, while in the other tumour, varying genetic imbalances along the long arm of chromosome 14 were detected. In summary, here, we report for the first time, the high-resolution a-CGH profiles of chromosome 14q in meningiomas, confirming that monosomy 14 is the most frequent alteration associated with this chromosome; other numerical abnormalities being only sporadically detected.

We describe a contact printing approach for microarrays that uses fused-silica capillary tubes with tapered tips for printing pins and a pressure/vacuum system to control pin loading, printing, and cleaning. The printing process is insensitive to variable environmental factors such as humidity, and the small diameter of the pins allows routine printing from 1536 well source plates. Pin load capacity, 0.2 microL in the current system, is adjustable by controlling pin length. More than 2000 spots can be printed per 0.2-microL pin load (<100 pl/spot), and densities of >12,000 spots/cm(2) are readily achievable. Solutions with a wide range of viscosities and chemical properties can be printed. The system can print tens of thousands of different solutions at high speed, due to the ability to use large numbers of pins simultaneously, and can produce a large number of replicate arrays since all of the solution picked up by the pins is available for deposition.

A peculiar adrenal tumor was analyzed using immunohistochemistry, electron microscopy, and fluorescence in situ hybridization (FISH) with multiple bacterial artificial chromosome (BAC) probes. The patient was a 34-year-old woman with a mass above the left kidney and multiple metastases. Her serum and urine dopamine level were elevated, and a diagnosis of malignant pheochromocytoma was made. The patient died approximately 3 years after her first visit. On post-mortem an adrenal tumor composed of small round cells forming Homer Wright rosette-like structures, a feature rarely observed in pheochromocytoma, was found. Immunohistochemistry was positive for chromogranin A and synaptophysin, and negative for cytokeratin, vimentin and neurofilaments. Because these results did not rule out a diagnosis of neuroblastoma, the tumor was further characterized on FISH with multiple BAC probes for loci known to be altered in neuroblastoma or pheochromocytoma, according to information in the literature that was for the most part obtained using comparative genomic hybridization. FISH demonstrated loss of heterozygosity at 11p, and gains at 16p, 19p, and 19q, a profile that favored a diagnosis of malignant pheochromocytoma over neuroblastoma. This case demonstrates that repeating FISH is useful for differential diagnosis.

Whole-genome sequencing of the soybean (Glycine max (L.) Merr. 'Williams 82') has made it important to integrate its physical and genetic maps. To facilitate this integration of maps, we screened 3290 microsatellites (SSRs) identified from BAC end sequences of clones comprising the 'Williams 82' physical map. SSRs were screened against 3 mapping populations. We found the AAT and ACT motifs produced the greatest frequency of length polymorphisms, ranging from 17.2% to 32.3% and from 11.8% to 33.3%, respectively. Other useful motifs include the dinucleotide repeats AG, AT, and AG, with frequency of length polymorphisms ranging from 11.2% to 18.4% (AT), 12.4% to 20.6% (AG), and 11.3% to 16.4% (GT). Repeat lengths less than 16 bp were generally less useful than repeat lengths of 40-60 bp. Two hundred and sixty-five SSRs were genetically mapped in at least one population. Of the 265 mapped SSRs, 60 came from BAC singletons not yet placed into contigs of the physical map. One hundred and ten originated in BACs located in contigs for which no genetic map location was previously known. Ninety-five SSRs came from BACs within contigs for which one or more other BACs had already been mapped. For these fingerprinted contigs (FPC) a high percentage of the mapped markers showed inconsistent map locations. A strategy is introduced by which physical and genetic map inconsistencies can be resolved using the preliminary 4x assembly of the whole genome sequence of soybean.

Human subtelomere regions contain numerous gene-rich segments and are susceptible to germline rearrangements. The availability of diagnostic test kits to detect subtelomeric rearrangements has resulted in the diagnosis of numerous abnormalities with clinical implications including congenital heart abnormalities and mental retardation. Several of these have been described as clinically recognizable syndromes (e.g., deletion of 1p, 3p, 5q, 6p, 9q, and 22q). Given this, fine-mapping of subtelomeric breakpoints is of increasing importance to the assessment of genotype-phenotype correlations in these recognized syndromes as well as to the identification of additional syndromes. We developed a BAC and cosmid-based DNA array (TEL array) with high-resolution coverage of 10 Mb-sized subtelomeric regions, and used it to analyze 42 samples from unrelated patients with subtelomeric rearrangements whose breakpoints were previously either unmapped or mapped at a lower resolution than that achievable with the TEL array. Six apparently recurrent subtelomeric breakpoint loci were localized to genomic regions containing segmental duplication, copy number variation, and sequence gaps. Small (1 Mb or less) candidate gene regions for clinical phenotypes in separate patients were identified for 3p, 6q, 9q, and 10p deletions as well as for a 19q duplication. In addition to fine-mapping nearly all of the expected breakpoints, several previously unidentified rearrangements were detected., ((c) 2008 Wiley-Liss, Inc.)

The latest revolution in the DNA sequencing field has been brought about by the development of automated sequencers that are capable of generating giga base pair data sets quickly and at low cost. Applications of such technologies seem to be limited to resequencing and transcript discovery, due to the shortness of the generated reads. In order to extend the fields of application to de novo sequencing, we developed the SHARCGS algorithm to assemble short-read (25-40-mer) data with high accuracy and speed. The efficiency of SHARCGS was tested on BAC inserts from three eukaryotic species, on two yeast chromosomes, and on two bacterial genomes (Haemophilus influenzae, Escherichia coli). We show that 30-mer-based BAC assemblies have N50 sizes >20 kbp for Drosophila and Arabidopsis and >4 kbp for human in simulations taking missing reads and wrong base calls into account. We assembled 949,974 contigs with length >50 bp, and only one single contig could not be aligned error-free against the reference sequences. We generated 36-mer reads for the genome of Helicobacter acinonychis on the Illumina 1G sequencing instrument and assembled 937 contigs covering 98% of the genome with an N50 size of 3.7 kbp. With the exception of five contigs that differ in 1-4 positions relative to the reference sequence, all contigs matched the genome error-free. Thus, SHARCGS is a suitable tool for fully exploiting novel sequencing technologies by assembling sequence contigs de novo with high confidence and by outperforming existing assembly algorithms in terms of speed and accuracy.

Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

Molecular confinement offers new routes for arraying large DNA molecules, enabling single-molecule schemes aimed at the acquisition of sequence information. Such schemes can rapidly advance to become platforms capable of genome analysis if elements of a nascent system can be integrated at an early stage of development. Integrated strategies are needed for surmounting the stringent experimental requirements of nanoscale devices regarding fabrication, sample loading, biochemical labeling, and detection. We demonstrate that disposable devices featuring both micro- and nanoscale features can greatly elongate DNA molecules when buffer conditions are controlled to alter DNA stiffness. Furthermore, we present analytical calculations that describe this elongation. We also developed a complementary enzymatic labeling scheme that tags specific sequences on elongated molecules within described nanoslit devices that are imaged via fluorescence resonance energy transfer. Collectively, these developments enable scaleable molecular confinement approaches for genome analysis.

Bacterial artificial chromosome (BAC) libraries are the large DNA insert libraries of choice and valuable tools for the map-based cloning of target quantitative trait loci, physical mapping, molecular cytogenetics and comparative genomics. The protocol reported here is a simplified method used to produce and screen BAC libraries from Brachypodium species and other related grasses. Intact nuclei, containing high molecular weight (HMW) DNA, are isolated and embedded in agarose plugs. The HMW DNA is digested using an appropriate restriction enzyme and size-fractionated using pulsed-field gel electrophoresis. The DNA is isolated by dialysis, ligated into pre-prepared vector and electroporated into competent Escherichia coli cells. A PCR-based method for screening the library is also described. The entire protocol takes at least 6 weeks to complete.

Background: Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project., Results: A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly., Conclusion: Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.

The karyotype of Indian muntjacs (Muntiacus muntjak vaginalis) has been greatly shaped by chromosomal fusion, which leads to its lowest diploid number among the extant known mammals. We present, here, comparative results based on draft sequences of 37 bacterial artificial clones (BAC) clones selected by chromosome painting for this special muntjac species. Sequence comparison on these BAC clones uncovered sequence syntenic relationships between the muntjac genome and those of other mammals. We found that the muntjac genome has peculiar features with respect to intron size and evolutionary rates of genes. Inspection of more than 80 pairs of orthologous introns from 15 genes reveals a significant reduction in intron size in the Indian muntjac compared to that of human, mouse, and dog. Evolutionary analysis using 19 genes indicates that the muntjac genes have evolved rapidly compared to other mammals. In addition, we identified and characterized sequence composition of the first BAC clone containing a chromosomal fusion site. Our results shed new light on the genome architecture of the Indian muntjac and suggest that chromosomal rearrangements have been accompanied by other salient genomic changes.

High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.