Large-insert DNA Library Research Articles

Construction of BAC Libraries from Flow-Sorted Chromosomes.

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

Preparation of sub-genomic fractions enriched for particular chromosomes in polyploid wheat

Flow-sorted chromosomes have been used to simplify analyses of complex plant genomes. In bread wheat, majority of studies involve cultivar Chinese Spring, a genotype chosen for sequencing. Telosomic lines developed from this cultivar enable isolation by flow sorting chromosome arms, which represent less than 3.4 % of the genome. However, access to other wheat cultivars is needed to allow mapping and cloning useful genes. In these cultivars, cytogenetic stocks are not readily available and only one chromosome (3B) can be sorted. Remaining chromosomes form composite peaks on flow karyotypes and cannot be sorted. In order to overcome this difficulty, we tested a pragmatic approach in which composite chromosome peaks are dissected to smaller sections. The analysis of chromosome composition in sorted fractions confirmed feasibility of obtaining sub-genomic fractions comprising only a few chromosomes. Usually one of the chromosomes was more abundant and the frequencies of dominant chromosomes in sorted fractions ranged from 16 % (chromosome 7B) to 80 % (chromosome 2B). The enrichment factor, calculated as the relative proportion of chromosomal DNA in the wheat genome to the proportion of chromosomal DNA in a sorted fraction, ranged from 3.2-fold (7B) to 16.4-fold (5D). At least a 5-fold enrichment can be obtained for 17 out of 21 wheat chromosomes. Moreover, we show that 15 out of the 21 chromosomes can be sorted without being contaminated by their homoeologs. These observations provide opportunities for constructing sub-genomic large-insert DNA libraries, optical mapping, and targeted sequencing selected genome regions in various cultivars of wheat. The availability of fractions enriched for chromosomes of interest and free of contaminating homoeologs will increase the efficiency of research projects and reduce their costs as compared to whole genome approaches. The same methodology should be feasible in other plants where single chromosome types cannot be sorted.

High-throughput physical map anchoring via BAC-pool sequencing

BackgroundPhysical maps created from large insert DNA libraries, typically cloned in BAC vector, are valuable resources for map-based cloning and de novo genome sequencing. The maps are most useful if contigs of overlapping DNA clones are anchored to chromosome(s), and ordered along them using molecular markers. Here we present a novel approach for anchoring physical maps, based on sequencing three-dimensional pools of BAC clones from minimum tilling path.ResultsWe used physical map of wheat chromosome arm 3DS to validate the method with two different DNA sequence datasets. The first comprised 567 genes ordered along the chromosome arm based on syntenic relationship of wheat with the sequenced genomes of Brachypodium, rice and sorghum. The second dataset consisted of 7,136 SNP-containing sequences, which were mapped genetically in Aegilops tauschii, the donor of the wheat D genome. Mapping of sequence reads from individual BAC pools to the first and the second datasets enabled unambiguous anchoring 447 and 311 3DS-specific sequences, respectively, or 758 in total.ConclusionsWe demonstrate the utility of the novel approach for BAC contig anchoring based on mass parallel sequencing of three-dimensional pools prepared from minimum tilling path of physical map. The existing genetic markers as well as any other DNA sequence could be mapped to BAC clones in a single in silico experiment. The approach reduces significantly the cost and time needed for anchoring and is applicable to any genomic project involving the construction of anchored physical map.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0429-1) contains supplementary material, which is available to authorized users.

Toward a Molecular Cytogenetic Map for Cultivated Sunflower (Helianthus annuus L.) by Landed BAC/BIBAC Clones

Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (∼100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC- fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)−derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources.

Construction and preliminary characterization of a river buffalo bacterial artificial chromosome library

River buffalo genome analyses have advanced significantly in the last decade, and the genome sequence of Bubalus bubalis will be available shortly. Nonetheless, large-insert DNA library resources such as bacterial artificial chromosomes (BAC) are still required for validation and accurate assembly of the genome sequence. We constructed a river buffalo BAC library containing 52,224 clones with an average insert size of 97 kb, representing 1.7 × coverage of the genome. This genomic resource for river buffalo will facilitate further studies in this economically important species allowing for instance, whole genome physical mapping and isolation of genes and gene clusters, contributing to the elucidation of gene organization and identification of regulatory elements.

A large insert Thellungiella halophila BIBAC library for genomics and identification of stress tolerance genes

Salt cress (Thellungiella halophila), a salt-tolerant relative of Arabidopsis, has turned to be an important model plant for studying abiotic stress tolerance. One binary bacterial artificial chromosome (BIBAC) library was constructed which represents the first plant-transformation-competent large-insert DNA library generated for Thellungiella halophila. The BIBAC library was constructed in BamHI site of binary vector pBIBAC2 by ligation of partial digested nuclear DNA of Thellungiella halophila. This library consists of 23,040 clones with an average insert size of 75 kb, and covers 4x Thellungiella halophila haploid genomes. BIBAC clones which contain inserts over 50 kb were selected and transformed into Arabidopsis for salt tolerant plant screening. One transgenic line was found to be more salt tolerant than wild type plants from the screen of 200 lines. It was demonstrated that the library contains candidates of stress tolerance genes and the approach is suitable for the transformation of stress susceptible plants for genetic improvement.

Splinkbes: A Splinkerette-Based Method for Generating Long end Sequences from Large Insert DNA Libraries

We report on the development of a novel splinkerette-based method for generating long end sequences from large insert library clones, using a carrot (Daucus carota L.) BAC library as a model. The procedure involves digestion of the BAC DNA with a 6-bp restriction enzyme, followed by ligation of splinkerette adaptors that serve as primer-annealing sites for PCR amplification of the BAC ends. The resulting amplicons are sequenced from both directions, and when overlapping, the pairs of sequences are assembled, originating two types of BAC end sequences (BESs): ungapped and gapped. The average sequence length for ungapped and gapped BESs was 698 and 1055 nucleotides, respectively, with an overall average length of 838 nucleotides. This is considerably higher than the average length typically obtained by direct end sequencing. Through the analysis of actual and in silico-generated BES of different lengths from carrot and five model organisms, we demonstrated that longer BESs are more informative, since they had more matches to the GenBank database and contained more simple sequence repeats (SSRs). A pilot high-throughput procedure is proposed for splinkerette-based end sequencing (SplinkBES). This method may contribute to generating more robust BES analysis and provide a richer source of BES-derived markers for genomics, mapping, and breeding.

Isolation and Cloning of High-Molecular-Weight Metagenomic DNA from Soil Microorganisms

INTRODUCTIONThe successful construction of large-insert community DNA (i.e., metagenomic) libraries from natural environments is dependent on several parameters, including effective cell lysis, DNA purity, and a high transformation efficiency. One problem associated with constructing metagenomic libraries from soil microbes is the co-isolation of contaminants, leading to the degradation of DNA as a result of nuclease activity. Because the isolation of intact genetic pathways from soil microbes is necessary to characterize their genetic and functional diversity, obtaining high-purity, high-molecular-weight (HMW) DNA for library construction is absolutely critical. This protocol describes the steps for the indirect extraction of bacterial DNA from soil, embedding the DNA in an agarose matrix, using a formamide and high-salt treatment to eliminate nucleases, size-selecting DNA by restriction digestion and pulsed-field gel electrophoresis (PFGE), and cloning the HMW DNA into a large-insert vector. The resulting metagenomic libraries contain high-purity, stable, HMW DNA that can be screened for various genetic loci (sequence-based) or phenotypic traits (function-based).

Construction of a BAC Library for a Defoliating Insect-Resistant Soybean and Identification of Candidate Clones Using a Novel Approach

Positional cloning of an insect-resistance quantitative trait locus (QTL) requires the construction of a large-insert genomic DNA library from insect-resistant genotypes. To facilitate cloning of a major defoliating insect-resistance QTL on linkage group M of the soybean genetic map, a bacterial artificial chromosome (BAC) library for PI 229358 was constructed and characterized. The HindIII BAC library contains 55,296 clones with an average insert size 131 kb. This library represents a 6-fold soybean haploid genome equivalents, allowing a 99.8% probability of recovering any specific sequence of interest in soybean. BAC filters were screened with a genomic DNA probe Sat_258sc2 obtained through genome walking from flanking sequences of a simple sequence repeat (SSR) marker, Sat_258, which links to the insect-resistance QTL. Thirteen BAC clones were identified positive for Sat_258sc2, and two of them were confirmed to carry Sat_258. The results suggest that this library is useful in positional cloning of the major insect-resistance QTL, and the approach presented here can be used to screen a BAC library for a SSR marker without requiring the creation of BAC pools.

The Complete Genome Sequence ofEscherichia coliDH10B: Insights into the Biology of a Laboratory Workhorse

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.

A machine-learning approach to combined evidence validation of genome assemblies

While it is common to refer to 'the genome sequence' as if it were a single, complete and contiguous DNA string, it is in fact an assembly of millions of small, partially overlapping DNA fragments. Sophisticated computer algorithms (assemblers and scaffolders) merge these DNA fragments into contigs, and place these contigs into sequence scaffolds using the paired-end sequences derived from large-insert DNA libraries. Each step in this automated process is susceptible to producing errors; hence, the resulting draft assembly represents (in practice) only a likely assembly that requires further validation. Knowing which parts of the draft assembly are likely free of errors is critical if researchers are to draw reliable conclusions from the assembled sequence data. We develop a machine-learning method to detect assembly errors in sequence assemblies. Several in silico measures for assembly validation have been proposed by various researchers. Using three benchmarking Drosophila draft genomes, we evaluate these techniques along with some new measures that we propose, including the good-minus-bad coverage (GMB), the good-to-bad-ratio (RGB), the average Z-score (AZ) and the average absolute Z-score (ASZ). Our results show that the GMB measure performs better than the others in both its sensitivity and its specificity for assembly error detection. Nevertheless, no single method performs sufficiently well to reliably detect genomic regions requiring attention for further experimental verification. To utilize the advantages of all these measures, we develop a novel machine learning approach that combines these individual measures to achieve a higher prediction accuracy (i.e. greater than 90%). Our combined evidence approach avoids the difficult and often ad hoc selection of many parameters the individual measures require, and significantly improves the overall precisions on the benchmarking data sets.

Construction of a 7-fold BAC library and cytogenetic mapping of 10 genes in the giant panda (Ailuropoda melanoleuca)

BackgroundThe giant panda, one of the most primitive carnivores, is an endangered animal. Although it has been the subject of many interesting studies during recent years, little is known about its genome. In order to promote research on this genome, a bacterial artificial chromosome (BAC) library of the giant panda was constructed in this study.ResultsThis BAC library contains 198,844 clones with an average insert size of 108 kb, which represents approximately seven equivalents of the giant panda haploid genome. Screening the library with 15 genes and 8 microsatellite markers demonstrates that it is representative and has good genome coverage. Furthermore, ten BAC clones harbouring AGXT, GHR, FSHR, IRBP, SOX14, TTR, BDNF, NT-4, LH and ZFX1 were mapped to 8 pairs of giant panda chromosomes by fluorescence in situ hybridization (FISH).ConclusionThis is the first large-insert genomic DNA library for the giant panda, and will contribute to understanding this endangered species in the areas of genome sequencing, physical mapping, gene cloning and comparative genomic studies. We also identified the physical locations of ten genes on their relative chromosomes by FISH, providing a preliminary framework for further development of a high resolution cytogenetic map of the giant panda.

Construction and characterization of a peanut HindIII BAC library

Bacterial artificial chromosome (BAC) libraries have been an essential tool for physical analyses of genomes of many crops. We constructed and characterized the first large-insert DNA library for Arachis hypogaea L. The HindIII BAC library contains 182,784 clones; only 5,484 (3%) had no inserts; and the average insert size is 104.05 kb. Chloroplast DNA contamination was very low, only nine clones, and r-DNA content was 1,208, 0.66% of clones. The depth of coverage is estimated to be 6.5 genome-equivalents, allowing the isolation of virtually any single-copy locus. This rate of coverage was confirmed with the application of 20 overgos, which identified 305 positive clones from the library. The identification of multiple loci by most probes in polyploids complicates anchoring of physical and genetic maps. We explored the practicality of a hybridization-based approach for determination of map locations of BAC clones in peanut by analyzing 94 clones detected by seven different overgos. The banding patterns on Southern blots were good predictors of contig composition; that is, the clones that shared the same size bands and ascribed to the same overgos usually also located in the same contigs. This BAC library has great potential to advance future research about the peanut genome.

High-resolution mapping and chromosome landing at the root-know nematode resistance locus Ma from Myrobalan plum using a large-insert BAC DNA library.

The Ma gene for root-knot nematode (RKN)resistance from Myrobalan plum (Prunus cerasifera L.)confers a complete-spectrum and a heat-stable resistance to Meloidogvne spp., conversely to Mi-I from tomato,which has a more restricted spectrum and a reduced efficiency at high temperature. This gene was identified from a perennial self-incompatible near-wild rootstock species and lies in cosegregation with the SCAR marker SCAFLP2 on the Prunus linkage group 7 in a 2.3 cM interval between the SCAR SCAL19 and SSR pchgms6 markers. We initiated a map-based cloning of Ma and report here the strategy that rapidly led to fine mapping and direct chromosome landing at the locus. Three pairs of bulks, totaling 90 individuals from half-sibling progenies derived from the Ma-heterozygous resistant accession P.2175, were constructed using mapping data, and saturation of the Ma region was performed by bulked segregant analysis (BSA) of 320 AFLP primer pair combinations. The closest three AFLP markers were transformed into codominant SCARs or CAPS designatedSCAFLP3, SCAFLP4 and SCAFLP5. By completing the mapping population up to 1,332 offspring from P.2175,Ma and SCAFLP2 were mapped in a 0.8 cM interval between SCAFLP3 and SCAFLP4. A large-insert bacterial artificial chromosome (BAC) DNA library of P.2175,totaling 30,720 clones with a mean insert size of 145 kb and a 14-15x Prunus haploid genome coverage was constructed and used to land on the Ma spanning interval with few BAC clones. As P.2175 is heterozygous for the gene, we constructed the resistant and susceptible physical contigs by PCR screening of the library with codominant markers. Additional microsatellite markers were then designed from BAC subcloning or BAC end sequencing. In the resistant contig, a single 280 kb BAC clone was shown to carry the Ma gene; this BAC contains two flanking markers on each side of the gene as well as two cosegregating markers. These results should allow future cloning of the Ma gene in this perennial species.

Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi).

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey (Ateles geoffroyi). A total of 193 152 BAC clones were generated in this library. The average insert size of the BAC clones was estimated to be 184.6 kb with the small inserts (50-100 kb) accounting for less than 3% and the non-recombinant clones only 1.2%. Assuming a similar genome size with humans, the spider monkey BAC library has about 11x genome coverage. In addition, by end sequencing of randomly selected BAC clones, we generated 367 sequence tags for the library. When blasted against human genome, they showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the library. This black-handed spider monkey BAC library would serve as a valuable resource in comparative genomic study and large-scale genome sequencing of nonhuman primates.

BAC end sequencing.

Large-insert genomic DNA libraries are based on the Escherichia coli F factor, a low-copy plasmid that exits in a supercoiled circular form in the host cells. These libraries are used to provide a way to divide complex genomes into DNA segments, thereby reducing the complexity (). The libraries are arrayed in microtiter dishes, providing the opportunity for many researchers to accumulate and use information regarding particular clones. The information about the end sequence of the bacterial artificial chromosome (BAC) clones is currently used in a wide array of applications from genome sequencing to gene discovery. The BAC end sequences of individual clones with insert DNA are collected in the large databases that can be accessed by the researcher. The information regarding the BAC end sequence stored in these databases makes it easier to identify the minimally overlapping clones that can be used as a source for shotgun-sequencing projects (), to find clones for restriction fingerprints for building overlapping clone sets (), to find appropriate clones for fluorescence in situ hybridization mapping (), or to select a BAC clone that contains genes of interest (). Some important BAC end sequence databases are as follows: 1. Human: http://www.tigr.org/tdb/humgen/bac_end_search/bac_end_search.html, 470,000 clones. 2. Rice: http://www.genome.clemson.edu/projects/rice/rice_bac_end/index.html, 92,000 clones. 3. Mouse: http://www.tigr.org/tdb/bac_ends/mouse/bac_end_intro.html, 300,000 clones. 4. Rat: http://www.tigr.org/tdb/bac_ends/rat/bac_end_intro.html, 200,000 clones. 5. Sea urchin: http://sugp.caltech.edu:7000, 25,000 clones. 6. Arabidopsis thaliana: http://ftp.tigr.org/tdb/at/abe/bac_end_search.html, >40,000 clones. 7. Trypanosoma brucei: http://ftp.tigr.org/tdb/mdb/tbdb/bac_end_search.html, 10,000 clones.

Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips

Simple, fast and cost-effective method for preparation of DNA with high molecular weight (HMW DNA) from plant nuclei and mitotic chromosomes has been developed. The technique involves mechanical homogenization of formaldehyde-fixed root tips, purification of nuclei and/or chromosomes on sucrose gradient, embedding in low-melting-point agarose, and DNA isolation in agarose plugs. Alternatively, nuclei and chromosomes may be purified using flow cytometry. Majority of DNA obtained is megabase-sized and well digestible by restriction endonucleases. The method is highly efficient as microgram amounts of DNA can be obtained from only several milligrams of plant tissue. Handling negligible amounts of plant material reduces the consumption of chemicals. Furthermore, the use of root tips makes it possible to obtain high-quality DNA even from plant species with leaves that are rigid or rich in secondary metabolites such as polyphenols. It is expected that preparation of HMW DNA from root tip nuclei will facilitate long-range mapping and construction of large-insert DNA libraries also in these species. Successful isolation of HMW DNA from flow-sorted chromosomes opens a way for construction of chromosome-specific large-insert libraries in plants.

Analysis and sorting of rye (Secale cerealeL.) chromosomes using flow cytometry

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.

Progress of Map-based Cloning of the Vf-resistance Gene and Functional Verification: Preliminary Results from Expression Studies in Transformed Apple

Progress of Map-based Cloning of the Vf-resistance Gene and Functional Verification: Preliminary Results from Expression Studies in Transformed Apple

One large-insert plant-transformation-competent BIBAC library and three BAC libraries of Japonica rice for genome research in rice and other grasses.

We report one large-insert BIBAC library and three BAC libraries for japonica rice cv Nipponbare. The BIBAC library was constructed in the HindIII site of a plant-transformation-competent binary vector (pCLD04541) and the three BAC libraries were constructed in the BamHI, HindIII and EcoRI sites of a BAC vector (pECBAC1), respectively. Each library contains 23,040 clones, has an average insert size of 130 kb, 170 kb, 150 kb and 156 kb, and covers 6.7x, 8.7x, 7.7x and 8.0 x rice haploid genomes, respectively. The combined libraries contain 92,160 clones in total, covering 31.1 x rice haploid genomes. To demonstrate their utility, we screened the libraries with 55 DNA markers mapped to chromosome 8 of the rice genetic maps and analyzed a number of clones by the restriction fingerprinting and contig assembly method. The results indicate that the libraries completely cover the rice genome and, thus, are well-suited for genome research in rice and other gramineous crops. The BIBAC library represents the first plant-transformation-competent large-insert DNA library for rice, which will streamline map-based cloning, functional analysis of the rice genome sequence and molecular breeding in rice and other grass species. These libraries are being used in the development of a whole-genome, BAC/BIBAC-based, integrated physical, genetic and sequence map of rice and in the research of genome-wide comparative genomics of grass species.