scholarly journals Utilization of Super BAC Pools and Fluidigm Access Array Platform for High-Throughput BAC Clone Identification: Proof of Concept

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Peter J. Maughan ◽  
Scott M. Smith ◽  
Joshua A. Raney
Keyword(s):  
Bac Library ◽  
Pcr Amplification ◽  
Artificial Chromosome ◽  
Nucleotide Polymorphisms ◽  
Bac Clone ◽  
Pcr Screening ◽  
Clone Identification ◽  
Bac Clones ◽  
Array Platform ◽  
Fluidigm Access Array

Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooledAmaranthus hypochondriacusBAC library. Ninety-six SNP loci, spanning the length ofA. hypochondriacuslinkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC). Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of~$0.05per reaction.

scholarly journals PCR identification of durum wheat BAC clones containing genes coding for carotenoid biosynthesis enzymes and their chromosome localization

Genome ◽  
2004 ◽  
Vol 47 (5) ◽  
pp. 911-917 ◽  
Author(s):  
A Cenci ◽  
S Somma ◽  
N Chantret ◽  
J Dubcovsky ◽  
A Blanco
Keyword(s):  
Tetraploid Wheat ◽  
Bac Library ◽  
Pcr Amplification ◽  
Carotenoid Biosynthesis ◽  
Quality Trait ◽  
Substitution Lines ◽  
D Genome ◽  
Pcr Screening ◽  
Bac Clones ◽  
Essential Components

Carotenoids are essential components in all plants. Their accumulation in wheat seed determines the endosperm colour, which is an important quality trait in wheat. In this study, we report the isolation of BAC clones containing genes coding for three different enzymes of the carotenoid biosynthesis pathway: phytoene synthase (PSY), phytoene desaturase (PDS), and ζ-carotene desaturase (ZDS). Primers were designed on the basis of wheat ESTs similar to the sequences of these three genes in other species, and used to screen a BAC library from Triticum turgidum var. durum (2n = 28, genomes AABB). Eight, six, and nine 384-well plates containing at least one positive clone were found for PSY, PDS, and ZDS, respectively. BACs selected for each of these genes were then divided in two groups corresponding to the A and B genomes of tetraploid wheat, based on differences in the length of the PCR amplification products, conformation-sensitive gel electrophoresis (CSGE), or cleavage amplification polymorphisms. Positive clones were then assigned to chromosomes using a set of D genome substitution lines in T. turgidum var. durum 'Langdon'. PSY clones were localized on chromosomes 5A and 5B, PDS on chromosomes 4A and 4B, and ZDS on chromosomes 2A and 2B. The strategies used for the PCR screening of large BAC libraries and for the differentiation of BAC clones from different genomes in a polyploid species are discussed.Key words: wheat, carotenoid biosynthesis, BAC.

scholarly journals A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library

2003 ◽  
Vol 69 (5) ◽  
pp. 2684-2691 ◽  
Author(s):  
Mark R. Liles ◽  
Brian F. Manske ◽  
Scott B. Bintrim ◽  
Jo Handelsman ◽  
Robert M. Goodman
Keyword(s):  
16S Rrna ◽  
Bac Library ◽  
Metagenomic Library ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acid Uptake ◽  
Direct Pcr ◽  
Bac Clone ◽  
Bac Clones

ABSTRACT We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, α-Proteobacteria, and CFB groups; greater representation among the β- and γ-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division.

Bacterial artificial chromosome clones randomly selected for sequencing reveal genomic differences between soybean cultivars

2018 ◽  
Vol 69 (2) ◽  
pp. 131
Author(s):  
Tingting He ◽  
Longshu Yang ◽  
Xianlong Ding ◽  
Linfeng Chen ◽  
Yanwei Li ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Reference Genome ◽  
De Novo ◽  
Gene Annotation ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Complete Sequence ◽  
Nucleotide Polymorphisms ◽  
Structural Variations ◽  
Bac Clones

This study pioneered the use of multiple technologies to combine the bacterial artificial chromosome (BAC) pooling strategy with high-throughput next- and third-generation sequencing technologies to analyse genomic difference. To understand the genetic background of the Chinese soybean cultivar N23601, we built a BAC library and sequenced 10 randomly selected clones followed by de novo assembly. Comparative analysis was conducted against the reference genome of Glycine max var. Williams 82 (2.0). Therefore, our result is an assessment of the reference genome. Our results revealed that 3517 single nucleotide polymorphisms (SNPs) and 662 insertion–deletions (InDels) occurred in ~1.2 Mb of the genomic region and that four of the 10 BAC clones contained 15 large structural variations (72 887 bp) compared with the reference genome. Gene annotation of the reference genome showed that Glyma.18g181000 was missing from the corresponding position of the 10 BAC clones. Additionally, there may be a problem with the assembly of some positions of the reference genome. Several gap regions in the reference genome could be supplemented by using the complete sequence of the 10 BAC clones. We believe that accurate and complete BAC sequence is a valuable resource that contributes to the completeness of the reference genome.

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

Genome ◽  
2004 ◽  
Vol 47 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Yaping Qian ◽  
Li Jin ◽  
Bing Su
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Large Scale ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Spider Monkey ◽  
Ateles Geoffroyi ◽  
Comparative Genomic ◽  
Average Insert Size ◽  
Bac Clones ◽  
Genomic Study

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 11× 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.Key words: black-handed spider monkeys, Ateles geoffroyi, BAC library.

scholarly journals Use of a Mycobacterium tuberculosisH37Rv Bacterial Artificial Chromosome Library for Genome Mapping, Sequencing, and Comparative Genomics

1998 ◽  
Vol 66 (5) ◽  
pp. 2221-2229 ◽  
Author(s):  
Roland Brosch ◽  
Stephen V. Gordon ◽  
Alain Billault ◽  
Thierry Garnier ◽  
Karin Eiglmeier ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Bacterial Artificial Chromosome ◽  
Genome Mapping ◽  
Sequence Data ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Excellent Correlation ◽  
Sequencing Project ◽  
Polysaccharide Biosynthesis ◽  
Bac Clones

ABSTRACT The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosisgenome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of ∼150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.

Construction of a hexaploid wheat (Triticum aestivumL.) bacterial artificial chromosome library for cloning genes for stripe rust resistance

Genome ◽  
2005 ◽  
Vol 48 (6) ◽  
pp. 1028-1036 ◽  
Author(s):  
P Ling ◽  
X M Chen
Keyword(s):  
Triticum Aestivum ◽  
Stripe Rust ◽  
Hexaploid Wheat ◽  
Rust Resistance ◽  
Puccinia Striiformis ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Stripe Rust Resistance ◽  
Insert Size ◽  
Bac Clones

A hexaploid wheat (Triticum aestivum L.) bacterial artificial chromosome (BAC) library was constructed for cloning Yr5 and other genes conferring resistance to stripe rust (Puccinia striiformis f. sp. tritici). Intact nuclei from a Yr5 near-isogenic line were used to isolate high molecular weight DNA, which was partially cleaved with HindIII and cloned into pECBAC1 and pIndigoBAC-5 vectors. The wheat BAC library consisted of 422 400 clones arrayed in 1100 micro-titer plates (each plate with 384 wells). Random sampling of 300 BAC clones indicated an average insert size of 140 kb, with a size range from 25 to 365 kb. Ninety percent of the clones in the library had an insert size greater than 100 kb and fewer than 5% of the clones did not contain inserts. Based on an estimated genome size of 15 966 Mb for hexaploid wheat, the BAC library was estimated to have a total coverage of 3.58× wheat genome equivalents, giving approximately 96% probability of identifying a clone representing any given wheat DNA sequence. Twelve BAC clones containing an Yr5 locus-specific marker (Yr5STS7/8) were successfully selected by PCR screening of 3-dimensional BAC pools. The results demonstrated that the T. aestivum BAC library is a valuable genomic resource for positional cloning of Yr5. The library also should be useful in cloning other genes for stripe rust resistance and other traits of interest in hexaploid wheat.Key words: BAC library, BAC pools, hexaploid wheat, Puccinia striiformis f. sp. tritici, resistance gene, stripe rust, Triticum aestivum.

scholarly journals Development of a sequence-based reference physical map of pea (Pisum sativum L.)

2019 ◽  
Author(s):  
Krishna Kishore Gali ◽  
Bunyamin Tar’an ◽  
Mohammed-Amin Madoui ◽  
Edwin van der Vossen ◽  
Jan van Oeveren ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Positional Cloning ◽  
Physical Map ◽  
Repetitive Sequences ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Bac Clones ◽  
Pooled Dna ◽  
Mapping Technology ◽  
Generation Sequencing

AbstractWhole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (~4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.

scholarly journals DECONVOLUTING BAC–GENE RELATIONSHIPS USING A PHYSICAL MAP

2008 ◽  
Vol 06 (03) ◽  
pp. 603-622
Author(s):  
YONGHUI WU ◽  
LAN LIU ◽  
TIMOTHY J. CLOSE ◽  
STEFANO LONARDI
Keyword(s):  
Physical Map ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Hybridization Experiment ◽  
Pooling Design ◽  
Bac Clones ◽  
Hybridization Data ◽  
A Genome ◽  
The Right ◽  
Combinatorial Pooling

Deconvolution of relationships between bacterial artificial chromosome (BAC) clones and genes is a crucial step in the selective sequencing of regions of interest in a genome. It often includes combinatorial pooling of unique probes obtained from the genes (unigenes), and screening of the BAC library using the pools in a hybridization experiment. Since several probes can hybridize to the same BAC, in order for the deconvolution to be achievable the pooling design has to be able to handle a large number of positives. As a consequence, smaller pools need to be designed, which in turn increases the number of hybridization experiments, possibly making the entire protocol unfeasible. We propose a new algorithm that is capable of producing high-accuracy deconvolution even in the presence of a weak pooling design, i.e. when pools are rather large. The algorithm compensates for the decrease of information in the hybridization data by taking advantage of a physical map of the BAC clones. We show that the right combination of combinatorial pooling and our algorithm not only dramatically reduces the number of pools required, but also successfully deconvolutes the BAC–gene relationships with almost perfect accuracy. Software is available on request from the first author.

scholarly journals Oncogenicity of Virulent Marek's Disease Virus Cloned as Bacterial Artificial Chromosomes

2004 ◽  
Vol 78 (23) ◽  
pp. 13376-13380 ◽  
Author(s):  
Lawrence Petherbridge ◽  
Andrew C. Brown ◽  
Susan J. Baigent ◽  
Ken Howes ◽  
Melanie A. Sacco ◽  
...  
Keyword(s):  
Disease Virus ◽  
Artificial Chromosome ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Wild Type Virus ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Bac Clone ◽  
Artificial Chromosomes ◽  
Bac Clones

ABSTRACT Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the US2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.

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