A compartmentalized approach to the assembly of physical maps

<p>Abstract</p> <p>Background</p> <p>Physical maps have been historically one of the cornerstones of genome sequencing and map-based cloning strategies. They also support marker assisted breeding and EST mapping. The problem of building a high quality physical map is computationally challenging due to unavoidable noise in the input fingerprint data.</p> <p>Results</p> <p>We propose a novel compartmentalized method for the assembly of high quality physical maps from fingerprinted clones. The knowledge of genetic markers enables us to group clones into clusters so that clones in the same cluster are more likely to overlap. For each cluster of clones, a local physical map is first constructed using FingerPrinted Contigs (FPC). Then, all the individual maps are carefully merged into the final physical map. Experimental results on the genomes of rice and barley demonstrate that the compartmentalized assembly produces significantly more accurate maps, and that it can detect and isolate clones that would induce "chimeric" contigs if used in the final assembly.</p> <p>Conclusion</p> <p>The software is available for download at <url>http://www.cs.ucr.edu/~sbozdag/assembler/</url></p

Global expression mapping of mammalian genomes

he aim of genome projects is to decipher all the information contained within the DNA of an organism and to study the way this information is processed in physiological processes. It is believed that more than 95% of the information content of the mammalian genome is represented in the protein coding sequences that make up only approximately 2% of the DNA sequence. Consequently much effort is being invested in the study of coding sequences in the form of cDNA analysis. This thesis is concerned with the development of a new strategy for a highly parallel approach to analyse entire cDNA libraries. The strategy is based upon generating sufficient sequence information to identify uniquely more than 100,000 cDNA clones by hybridisation with short oligonucleotides, typically 7 - 10 mers. Each oligonucleotide is hybridised to all cDNA clones in parallel and under stringent conditions positively identifies a subset (3 - 10%) of clones. Oligonucleotides are designed in such a way that each will positively identify a different subset of clones and statistical simulations estimate that approximately 200 such hybridisation events are required to identify uniquely upto 100,000 cDNA sequences. Such a fingerprint can be generated from many cDNA libraries constructed from different tissue mRNAs and will not only lead to the identification of most sequecnes expressed from the genome but also indicate the level of expression by determining the number of times any given sequence is represented across different cDNA libraries. A human foetal brain cDNA library has been constructed and 100,000 clones arrayed into microtitre plates and on nylon membranes. All the required technological developments have been carried out successfully and are presented. In excess of 200 oligonucleotide hybridisations have been performed on a subset of 32,000 cDNA clones and 1,000 sequenced control clones. A detailed analysis of the data on the control clones is presented and the implications for cDNA fingerprinting discussed

A high-throughput strategy for screening of bacterial artificial chromosome libraries and anchoring of clones on a genetic map constructed with single nucleotide polymorphisms

<p>Abstract</p> <p>Background</p> <p>Current techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate™ assay.</p> <p>Results</p> <p>To test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 <it>Aegilops tauschii </it>BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an <it>Ae. tauschii </it>genetic map. Of 1384 allele-informative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted <it>Ae. tauschii </it>BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the <it>Ae. tauschii </it>genetic map. For 41 (95%) assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5%) assays, positive clones were in two different contigs and the relationships of these contigs to loci on the <it>Ae. tauschii </it>genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools.</p> <p>Conclusion</p> <p>The highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high-throughput anchoring of BAC contigs on genetic maps during the construction of physical maps of eukaryotic genomes. In most cases, screening of BAC libraries with Illumina oligonucleotide assays results in the unequivocal relationship of BAC clones with loci on the genetic map.</p

Assessing Plant Genetic Diversity by Molecular Tools

This paper is an overview of the diverse, predominantly molecular techniques, used in assessing plant genetic diversity. In recent years, there has been a significant increase in the application of molecular genetic methods for assessing the conservation and use of plant genetic resources. Molecular techniques have been applied in the analysis of specific genes, as well as to increase understanding of gene action, generate genetic maps and assist in the development of gene transfer technologies. Molecular techniques have also had critical roles in studies of phylogeny and species evolution, and have been applied to increase our understanding of the distribution and extent of genetic variation within and between species. These techniques are well established and their advantages as well as limitations have been realized and described in this work. Recently, a new class of advanced techniques has emerged, primarily derived from a combination of earlier, more basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques, in order to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. Some of the advanced marker techniques utilize newer classes of DNA elements, such as retrotransposons, mitochondrial and chloroplast based microsatellites, thereby revealing genetic variation through increased genome coverage. Techniques such as RAPD and AFLP are also being applied to cDNA-based templates to study patterns of gene expression and uncover the genetic basis of biological responses. The most important and recent advances made in molecular marker techniques are discussed in this review, along with their applications, advantages and limitations applied to plant sciences

A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening

<p>Abstract</p> <p>Background</p> <p>A five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones.</p> <p>Results</p> <p>A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~2× genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on <it>Aegilops tauschii </it>chromosome 2D and <it>Ae. tauschii </it>contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision.</p> <p>Conclusion</p> <p>A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at <url>http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml</url>.</p

A first generation BAC-based physical map of the channel catfish genome

BACKGROUND: Channel catfish, Ictalurus punctatus, is the leading species in North American aquaculture. Genetic improvement of catfish is performed through selective breeding, and genomic tools will help improve selection efficiency. A physical map is needed to integrate the genetic map with the karyotype and to support fine mapping of phenotypic trait alleles such as Quantitative Trait Loci (QTL) and the effective positional cloning of genes. RESULTS: A genome-wide physical map of the channel catfish was constructed by High-Information-Content Fingerprinting (HICF) of 46,548 Bacterial Artificial Chromosomes (BAC) clones using the SNaPshot technique. The clones were assembled into contigs with FPC software. The resulting assembly contained 1,782 contigs and covered an estimated physical length of 0.93 Gb. The validity of the assembly was demonstrated by 1) anchoring 19 of the largest contigs to the microsatellite linkage map 2) comparing the assembly of a multi-gene family to Restriction Fragment Length Polymorphism (RFLP) patterns seen in Southern blots, and 3) contig sequencing. CONCLUSION: This is the first physical map for channel catfish. The HICF technique allowed the project to be finished with a limited amount of human resource in a high throughput manner. This physical map will greatly facilitate the detailed study of many different genomic regions in channel catfish, and the positional cloning of genes controlling economically important production traits

Applications of molecular markers in genetic analysis

Restriction fragment length polymorphism (RFLP) and microsatellite molecular markers were used to map two soybean nodulation genes, enod2 and leghemoglobin (lbc3). In addition, a high annealing temperature DNA amplification fingerprinting (DAF) method was developed for DNA fingerprinting of soybean cyst nematode (SCN), Mychorrizae, aphid, centipedegrass, and bermudagrass samples. Recombinant inbred lines (RILs) as well as an F\u3c sub \u3e2 segregating population of soybean Glycine max (L. Merr) facilitated the mapping of two expressed sequence tags (EST) involved in early nodulation and subsequent nitrogen fixation in soybean. For the early nodulin gene enodl2, the parents of RILs, Minsoy and Noirl, showed a polymorphism (5.5 vs. 5.9 kb) after EcoRV digestion. RFLP patterns of 42 RILs were analyzed using the MAPMAKER program linking enod2 to the seed coat color gene, I, with a distance of 11.1 cM on linkage group U3 of RIL map. Enodl2 and I are located close to Rhg4 , a soybean cyst nematode (SCN) resistance gene, and a locus for seed coat hardness. The molecular marker pAllO and seed coat color were used to integrate enod2 on an F2 segregating population (72 plants) generated from a cross between cultivar Bragg and G. soja (Sieb and Zucc), PI468.397. Enod2 was mapped in the same order as on the RIL map but 18.5 cM from the I locus. A microsatellite from the 5\u27 region of enod2B was mapped in the same position, demonstrating that enod2B and not enod2A was mapped. An RFLP for lbc3 (leghemoglobin) segregated independently from enod2 and the nts-l supemodulating locus suggesting that in soybean, symbiotically significant loci (including rj1, rj2, and rj6) are not clustered. To overcome potential problems caused by mismatch priming and secondary DNA structure and taking advantage of high primer-template ratios used in DAF reactions, annealing temperature of 55°C were used with single short arbitrary oligonucleotide as well as mini-hairpin primers to provide high resolution DNA profiles of soybean. Initially, high annealing temperatures for three arbitrary octamer primers in polymerase chain reaction (PGR) were tested for DNA fingerprinting of two soybean cultivars, Minsoy and Noirl. Fifteen PGR programs differing in levels of annealing temperature (47, 55, and 60°C), denaturation, annealing, and extension time (30, 60, and 120 second), and presence/ absence of extension step (+/- 72°C) were tested. The number of bands (amplification products) ranged from 7 (Program 10) to 51 (Program 3). The average ramping temperature for heating and cooling were calculated 1.42 and 1.27 sec/°C, respectively. Intensity of the silver-stained bands in a 10% polyacrylamide gel was high for the most PCR programs. Program 15, DAF-15, (95°C/30 sec, 55°C/120, and 72°C/30 sec) generated a complex DNA fingerprinting profiles for tested primers in Minsoy and Noirl. These profiles contained an average of 42 sharp and highly intense bands using both octamer primers 8-4 and 8-8 for DNA amplification. Using high annealing temperature increased stringency of primer-template annealing, avoided potential mismatching and hybrid molecule formation, and consequently improved reproducibility of DNA fingerprinting. Newly-developed high annealing temperature DAF was used successfully and detected markers linked to the enod2 gene and analyzed DNA fingerprinting of soybean cyst nematode (SCN), Mycorrhizae, aphid, centipedegrass, and bermudagrass samples. RFLP patterns of 41 homozygous F2 individuals for enod2 gene were set into two bulks of 26 and 15 with RFLP patterns identical to their parental patterns Bragg and G. soja, respectively. Screening of the bulks B and S with 31 primers resulted in detection of four polymorphic bands using primers HpC29 and HpC30 and DAF-15 program. Due to low number of polymorphic bands in the B and S bulks, sub-pools were generated and screened. B1 and SI sub-pools were tested with total 196 primers of which 32 were used for screening of sub-pools B3 versus S2. Primers Hp30, HpC22 and HpC30 generated 1, 1 and 4 polymorphic markers, respectively, in the B3 vs. S2. The major screening was focused on the B1 versus SI sub-pools which resulted in screening of 196 mini-hairpin and unstructured primers of which a set of 9 primers detected 20 polymorphic bands. Primer HpD25 generated polymorphic bands with 920B1, 320B1, 220S1, and 185B1 base pairs which were reliable and reproducible. These bands are promising bands for further analysis such as cloning and generating SCAR markers in the region of genome containing the enod2 gene. Key Words: nitrogen fixation, RFLP, recombinant inbred lines, integration mapping, annealing temperature, PCR, DNA fingerprinting, arbitrary primers, soybean. Abbreviations: RFLP, restriction fragment length polymorphism; SCN, soybean cyst nematode; RJLs, recombinant inbred lines; CHS, chalcone synthase; QTL, quantitative trait locus; lbc3, leghemoglobin gene; DAF, DNA amplification fingerprinting; PCR, polymerase chain reaction

The human Y chromosome; Towards a physical map in terms of overlapping cosmid clones

This thesis describes the construction and analysis of a cosmid library specific for the human Y chromosome. The purpose of preparing such a library is to provide a source of ordered overlapping clones for use in the identification of genes, analysis of chromosome structure, and perhaps, as a starting point for the sequencing of the chromosome. Approximately 2,000 cosmid clones containing inserts derived from human DNA have been identified in a library prepared from a somatic cell hybrid containing the Y chromosome as the only human material. These clones have been analysed in two principal ways. Firstly, hybridisation to various characterised DNA fragments has identified cosmids containing known repeat elements and single copy DNA sequences which have been previously assigned to regions of the chromosome by deletion mapping. Secondly, a unique 'fingerprint' has been produced for each clone; band patterns of Hint I digestion products separated by polyacrylamiae gel electrophoresis have been analysed by computer to detect overlapping clones. A significant proportion of the chromosome is represented in this way