C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries.

Studies performed using Hi-C and other high-throughput whole-genome C-methods have demonstrated that 3D organization of eukaryotic genomes is functionally relevant. Unfortunately, ultra-deep sequencing of Hi-C libraries necessary to detect loop structures in large vertebrate genomes remains rather expensive. However, many studies are in fact aimed at determining the fine-scale 3D structure of comparatively small genomic regions up to several Mb in length. Such studies typically focus on the spatial structure of domains of coregulated genes, molecular mechanisms of loop formation, and interrogation of functional significance of GWAS-revealed polymorphisms. Therefore, a handful of molecular techniques based on Hi-C have been developed to address such issues. These techniques commonly rely on in-solution hybridization of Hi-C/3C-seq libraries with pools of biotinylated baits covering the region of interest, followed by deep sequencing of the enriched library. Here, we describe a new protocol of this kind, C-TALE (Chromatin TArget Ligation Enrichment). Preparation of hybridization probes from bacterial artificial chromosomes and an additional round of enrichment make C-TALE a cost-effective alternative to existing many-versus-all C-methods.

[Clinical Application Assessment of KaryoLite BoBs Combined with QF-PCR in the Detection of Products of Conception].

OBJECTIVE: To assess the accuracy and discuss the feasibility of KaryoLite bacterial artificial chromosome on beads (KL-BoBs) and quantitative fluorescent polymerase chain reaction (QF-PCR) in genetic testing of products of conception (POC) by comparing with the chromosomal microarray analysis (CMA) test results. METHODS: Eighty-one cases of abortion samples were collected in the prenatal diagnosis center of West China Second University Hospital in Sichuan University from May to August 2016,including 61 cases of placenta tissues,19 cases of fetal muscle tissues and 1 case of fetal liver tissue. KL-BoBs and QF-PCR were used to detect the samples. The results were compared with those of CMA test to evaluate the accuracy of KL-BoBs and QF-PCR. RESULTS: Of the 81 POC samples,the results of 70 samples tested by KL-BoBs was consistent with that of CMA. Among them,36 cases were normal karyotype and 34 cases were abnormal karyotypes (aneuploidy). Triploid could not been detected by KL-BoBs (the results were shown 2 cases were normal karyotype and 5 cases were aneuploidy),whereas CMA and QF-PCR could be detected. Copy number variation of small segments could not been detected by KL-Bobs. Four cases of copy number variationwere detected by CMA.Compared with CMA,the positive coincident rate of KL-BoBs combined with QF-PCR was 91.1% (41/45),the negative coincidence rate was 100% (36/36). The accuracy rate of KL-BoBs was 86.4% (70/81),the false positive was 0% and the false negative was 13.3% (6/45). Whereas both KL-BoBs and QF-PCR were simultaneously detected,the accuracy rate would be improved to 95.1% (77/81). CONCLUSION: The accuracy rate of KL-BoBs combined with QF-PCR was high for testing early pregnancy abortion tissue. It can be used as a first-tier test.

Independent assessment and improvement of wheat genome sequence assemblies using Fosill jumping libraries.

Background: The accurate sequencing and assembly of very large, often polyploid, genomes remains a challenging task, limiting long-range sequence information and phased sequence variation for applications such as plant breeding. The 15-Gb hexaploid bread wheat (Triticum aestivum) genome has been particularly challenging to sequence, and several different approaches have recently generated long-range assemblies. Mapping and understanding the types of assembly errors are important for optimising future sequencing and assembly approaches and for comparative genomics. Results: Here we use a Fosill 38-kb jumping library to assess medium and longer-range order of different publicly available wheat genome assemblies. Modifications to the Fosill protocol generated longer Illumina sequences and enabled comprehensive genome coverage. Analyses of two independent Bacterial Artificial Chromosome (BAC)-based chromosome-scale assemblies, two independent Illumina whole genome shotgun assemblies, and a hybrid Single Molecule Real Time (SMRT-PacBio) and short read (Illumina) assembly were carried out. We revealed a surprising scale and variety of discrepancies using Fosill mate-pair mapping and validated several of each class. In addition, Fosill mate-pairs were used to scaffold a whole genome Illumina assembly, leading to a 3-fold increase in N50 values. Conclusions: Our analyses, using an independent means to validate different wheat genome assemblies, show that whole genome shotgun assemblies based solely on Illumina sequences are significantly more accurate by all measures compared to BAC-based chromosome-scale assemblies and hybrid SMRT-Illumina approaches. Although current whole genome assemblies are reasonably accurate and useful, additional improvements will be needed to generate complete assemblies of wheat genomes using open-source, computationally efficient, and cost-effective methods.

MaGuS: a tool for quality assessment and scaffolding of genome assemblies with Whole Genome Profiling™ Data.

BACKGROUND: Scaffolding is an essential step in the genome assembly process. Current methods based on large fragment paired-end reads or long reads allow an increase in contiguity but often lack consistency in repetitive regions, resulting in fragmented assemblies. Here, we describe a novel tool to link assemblies to a genome map to aid complex genome reconstruction by detecting assembly errors and allowing scaffold ordering and anchoring. RESULTS: We present MaGuS (map-guided scaffolding), a modular tool that uses a draft genome assembly, a Whole Genome Profiling™ (WGP) map, and high-throughput paired-end sequencing data to estimate the quality and to enhance the contiguity of an assembly. We generated several assemblies of the Arabidopsis genome using different scaffolding programs and applied MaGuS to select the best assembly using quality metrics. Then, we used MaGuS to perform map-guided scaffolding to increase contiguity by creating new scaffold links in low-covered and highly repetitive regions where other commonly used scaffolding methods lack consistency. CONCLUSIONS: MaGuS is a powerful reference-free evaluator of assembly quality and a WGP map-guided scaffolder that is freely available at https://github.com/institut-de-genomique/MaGuS. Its use can be extended to other high-throughput sequencing data (e.g., long-read data) and also to other map data (e.g., genetic maps) to improve the quality and the contiguity of large and complex genome assemblies.

Genome-wide assessment of differential effector gene use in embryogenesis.

Six different populations of cells were isolated by fluorescence-activated cell sorting from disaggregated late blastula- and gastrula-stage sea urchin embryos according to the regulatory states expressed in these cells, as reported by recombineered bacterial artificial chromosomes producing fluorochromes. Transcriptomes recovered from these embryonic cell populations revealed striking, early differential expression of large cohorts of effector genes. The six cell populations were presumptive pigment cells, presumptive neurogenic cells, presumptive skeletogenic cells, cells from the stomodeal region of the oral ectoderm, ciliated band cells and cells from the endoderm/ectoderm boundary that will give rise both to hindgut and to border ectoderm. Transcriptome analysis revealed that each of these domains specifically expressed several hundred effector genes at significant levels. Annotation indicated the qualitative individuality of the functional nature of each cell population, even though they were isolated from embryos only 1-2 days old. In no case was more than a tiny fraction of the transcripts enriched in one population also enriched in any other of the six populations studied. As was particularly clear in the cases of the presumptive pigment, neurogenic and skeletogenic cells, all three of which represent precociously differentiating cell types of this embryo, most specifically expressed genes of given cell types are not significantly expressed at all in the other cell types. Thus, at the effector gene level, a dramatic, cell type-specific pattern of differential gene regulation is established well before any significant embryonic morphogenesis has occurred.

Marked difference in conformational fluctuation between giant DNA molecules in circular and linear forms.

We performed monomolecular observations on linear and circular giant DNAs (208 kbp) in an aqueous solution by the use of fluorescence microscopy. The results showed that the degree of conformational fluctuation in circular DNA was ca. 40% less than that in linear DNA, although the long-axis length of circular DNA was only 10% smaller than that of linear DNA. Additionally, the relaxation time of a circular chain was shorter than that of a linear chain by at least one order of magnitude. The essential features of this marked difference between linear and circular DNAs were reproduced by numerical simulations on a ribbon-like macromolecule as a coarse-grained model of a long semiflexible, double-helical DNA molecule. In addition, we calculated the radius of gyration of an interacting chain in a circular form on the basis of the mean field model, which provides a better understanding of the present experimental trend than a traditional theoretical equation.

BAC chromosomal microarray for prenatal detection of chromosome anomalies in fetal ultrasound anomalies: an economic evaluation.

INTRODUCTION: To determine the cost-effectiveness of prenatal chromosomal microarray (CMA) when performed for structural anomalies on fetal ultrasound scan over conventional techniques. METHOD: A decision tree was populated using data from a prospective cohort of women undergoing testing when a fetal ultrasound scan showed a structural abnormality. Nine strategies of testing were modeled including combinations of the tests: QFPCR, G-band karyotyping, CMA and FISH for DiGeorge (22q) microdeletion. RESULTS: When CMA costs GBP 405 and using a 1-Mb BAC array it would cost GBP 24,600 for every additional case detected by CMA over a combination of QFPCR, followed by G-band karyotype, followed lastly by FISH (for DiGeorge syndrome). If CMA is performed instead of conventional karyotyping alone it costs GBP 33,000 for every additional case detected. However, if the cost of CMA is reduced to GBP 360 than when CMA is performed instead of conventional karyotyping alone it would cost GBP 9,768 for every additional case detected. DISCUSSION: The use of a prenatal BAC CMA is not currently cost-effective when compared to other testing strategies. However, as CMA costs decrease and resolution (and detection rates) increase it is likely to become the cost-effective option of the future.

A higher mutational burden in females supports a "female protective model" in neurodevelopmental disorders.

Increased male prevalence has been repeatedly reported in several neurodevelopmental disorders (NDs), leading to the concept of a "female protective model." We investigated the molecular basis of this sex-based difference in liability and demonstrated an excess of deleterious autosomal copy-number variants (CNVs) in females compared to males (odds ratio [OR] = 1.46, p = 8 × 10(-10)) in a cohort of 15,585 probands ascertained for NDs. In an independent autism spectrum disorder (ASD) cohort of 762 families, we found a 3-fold increase in deleterious autosomal CNVs (p = 7 × 10(-4)) and an excess of private deleterious single-nucleotide variants (SNVs) in female compared to male probands (OR = 1.34, p = 0.03). We also showed that the deleteriousness of autosomal SNVs was significantly higher in female probands (p = 0.0006). A similar bias was observed in parents of probands ascertained for NDs. Deleterious CNVs (>400 kb) were maternally inherited more often (up to 64%, p = 10(-15)) than small CNVs < 400 kb (OR = 1.45, p = 0.0003). In the ASD cohort, increased maternal transmission was also observed for deleterious CNVs and SNVs. Although ASD females showed higher mutational burden and lower cognition, the excess mutational burden remained, even after adjustment for those cognitive differences. These results strongly suggest that females have an increased etiological burden unlinked to rare deleterious variants on the X chromosome. Carefully phenotyped and genotyped cohorts will be required for identifying the symptoms, which show gender-specific liability to mutational burden.

Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment.

Over the past decade, major leaps forward have been made on the mechanistic understanding and identification of adaptive stress response landscapes underlying toxic insult using transcriptomics approaches. However, for predictive purposes of adverse outcome several major limitations in these approaches exist. First, the limited number of samples that can be analyzed reduces the in depth analysis of concentration-time course relationships for toxic stress responses. Second these transcriptomics analysis have been based on the whole cell population, thereby inevitably preventing single cell analysis. Third, transcriptomics is based on the transcript level, totally ignoring (post)translational regulation. We believe these limitations are circumvented with the application of high content analysis of relevant toxicant-induced adaptive stress signaling pathways using bacterial artificial chromosome (BAC) green fluorescent protein (GFP) reporter cell-based assays. The goal is to establish a platform that incorporates all adaptive stress pathways that are relevant for toxicity, with a focus on drug-induced liver injury. In addition, cellular stress responses typically follow cell perturbations at the subcellular organelle level. Therefore, we complement our reporter line panel with reporters for specific organelle morphometry and function. Here, we review the approaches of high content imaging of cellular adaptive stress responses to chemicals and the application in the mechanistic understanding and prediction of chemical toxicity at a systems toxicology level.

Accuracy and coverage assessment of Oryctolagus cuniculus (rabbit) genes encoding immunoglobulins in the whole genome sequence assembly (OryCun2.0) and localization of the IGH locus to chromosome 20.

We report on the analyses of genes encoding immunoglobulin heavy and light chains in the rabbit 6.51× whole genome assembly. This OryCun2.0 assembly confirms previous mapping of the duplicated IGK1 and IGK2 loci to chromosome 2 and the IGL lambda light chain locus to chromosome 21. The most frequently rearranged and expressed IGHV1 that is closest to IG DH and IGHJ genes encodes rabbit VHa allotypes. The partially inbred Thorbecke strain rabbit used for whole-genome sequencing was homozygous at the IGK but heterozygous with the IGHV1a1 allele in one of 79 IGHV-containing unplaced scaffolds and IGHV1a2, IGHM, IGHG, and IGHE sequences in another. Some IGKV, IGLV, and IGHA genes are also in other unplaced scaffolds. By fluorescence in situ hybridization, we assigned the previously unmapped IGH locus to the q-telomeric region of rabbit chromosome 20. An approximately 3-Mb segment of human chromosome 14 including IGH genes predicted to map to this telomeric region based on synteny analysis could not be located on assembled chromosome 20. Unplaced scaffold chrUn0053 contains some of the genes that comparative mapping predicts to be missing. We identified discrepancies between previous targeted studies and the OryCun2.0 assembly and some new BAC clones with IGH sequences that can guide other studies to further sequence and improve the OryCun2.0 assembly. Complete knowledge of gene sequences encoding variable regions of rabbit heavy, kappa, and lambda chains will lead to better understanding of how and why rabbits produce antibodies of high specificity and affinity through gene conversion and somatic hypermutation.

SeqEntropy: genome-wide assessment of repeats for short read sequencing.

BACKGROUND: Recent studies on genome assembly from short-read sequencing data reported the limitation of this technology to reconstruct the entire genome even at very high depth coverage. We investigated the limitation from the perspective of information theory to evaluate the effect of repeats on short-read genome assembly using idealized (error-free) reads at different lengths. METHODOLOGY/PRINCIPAL FINDINGS: We define a metric H(k) to be the entropy of sequencing reads at a read length k and use the relative loss of entropy ΔH(k) to measure the impact of repeats for the reconstruction of whole-genome from sequences of length k. In our experiments, we found that entropy loss correlates well with de-novo assembly coverage of a genome, and a score of ΔH(k)>1% indicates a severe loss in genome reconstruction fidelity. The minimal read lengths to achieve ΔH(k)<1% are different for various organisms and are independent of the genome size. For example, in order to meet the threshold of ΔH(k)<1%, a read length of 60 bp is needed for the sequencing of human genome (3.2 10(9) bp) and 320 bp for the sequencing of fruit fly (1.8×10(8) bp). We also calculated the ΔH(k) scores for 2725 prokaryotic chromosomes and plasmids at several read lengths. Our results indicate that the levels of repeats in different genomes are diverse and the entropy of sequencing reads provides a measurement for the repeat structures. CONCLUSIONS/SIGNIFICANCE: The proposed entropy-based measurement, which can be calculated in seconds to minutes in most cases, provides a rapid quantitative evaluation on the limitation of idealized short-read genome sequencing. Moreover, the calculation can be parallelized to scale up to large euakryotic genomes. This approach may be useful to tune the sequencing parameters to achieve better genome assemblies when a closely related genome is already available.

Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant.

The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

Microarray analysis of copy number variation in single cells.

We present a protocol for reliably detecting DNA copy number aberrations in a single human cell. Multiple displacement-amplified DNAs of a cell are hybridized to a 3,000-bacterial artificial chromosome (BAC) array and to an Affymetrix 250,000 (250K)-SNP array. Subsequent copy number calling is based on the integration of BAC probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGA) reference model for diploid chromosomes, as well as SNP copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). All methods for detecting DNA copy number aberrations in single human cells have difficulty in confidently discriminating WGA artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental DNA imbalance detection. Our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. Additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGA samples. The protocol takes 5-7 d.

Assessment of infectivity using a Herpesvirus saimiri (HVS) recombinant that expresses HVS-GFP.

Herpesvirus saimiri (HVS) is capable of infecting a wide range of human cell types with high efficiency. The viral genome persists as high-copy-number, circular, nonintegrated episomes that segregate to progeny on cell division. This allows HVS-based vectors to transduce stably a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome (BAC) cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. These properties offer characteristics similar to that of an artificial chromosome combined with an efficient delivery system. This protocol describes the use of an HVS recombinant virus expressing green fluorescent protein (GFP) (HVS-GFP) to assess the infectivity of a specific cell line.

Analysis of BAC-end sequences in rainbow trout: content characterization and assessment of synteny between trout and other fish genomes.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) are cultivated worldwide for aquaculture production and are widely used as a model species to gain knowledge of many aspects of fish biology. The common ancestor of the salmonids experienced a whole genome duplication event, making extant salmonids such as the rainbow trout an excellent model for studying the evolution of tetraploidization and re-diploidization in vertebrates. However, the lack of a reference genome sequence hampers research progress for both academic and applied purposes. In order to enrich the genomic tools already available in this species and provide further insight on the complexity of its genome, we sequenced a large number of rainbow trout BAC-end sequences (BES) and characterized their contents. RESULTS: A total of 176,485 high quality BES, were generated, representing approximately 4% of the trout genome. BES analyses identified 6,848 simple sequence repeats (SSRs), of which 3,854 had high quality flanking sequences for PCR primers design. The first rainbow trout repeat elements database (INRA RT rep1.0) containing 735 putative repeat elements was developed, and identified almost 59.5% of the BES database in base-pairs as repetitive sequence. Approximately 55% of the BES reads (97,846) had more than 100 base pairs of contiguous non-repetitive sequences. The fractions of the 97,846 non-repetitive trout BES reads that had significant BLASTN hits against the zebrafish, medaka and stickleback genome databases were 15%, 16.2% and 17.9%, respectively, while the fractions of the non-repetitive BES reads that had significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 10.7%, 9.5% and 9.5%, respectively. Comparative genomics using paired BAC-ends revealed several regions of conserved synteny across all the fish species analyzed in this study. CONCLUSIONS: The characterization of BES provided insights on the rainbow trout genome. The discovery of specific repeat elements will facilitate analyses of sequence content (e.g. for SNPs discovery and for transcriptome characterization) and future genome sequence assemblies. The numerous microsatellites will facilitate integration of the linkage and physical maps and serve as valuable resource for fine mapping QTL and positional cloning of genes affecting aquaculture production traits. Furthermore, comparative genomics through BES can be used for identifying positional candidate genes from QTL mapping studies, aid in future assembly of a reference genome sequence and elucidating sequence content and complexity in the rainbow trout genome.

High performance gel imaging with a commercial single lens reflex camera.

A high performance gel imaging system was constructed using a digital single lens reflex camera with epi-illumination to image 19 × 23 cm agarose gels with up to 10,000 DNA bands each. It was found to give equivalent performance to a laser scanner in this high throughput DNA fingerprinting application using the fluorophore SYBR Green(®). The specificity and sensitivity of the imager and scanner were within 1% using the same band identification software. Low and high cost color filters were also compared and it was found that with care, good results could be obtained with inexpensive dyed acrylic filters in combination with more costly dielectric interference filters, but that very poor combinations were also possible. Methods for determining resolution, dynamic range, and optical efficiency for imagers are also proposed to facilitate comparison between systems.

Analysis of BAC-end sequences (BESs) and development of BES-SSR markers for genetic mapping and hybrid purity assessment in pigeonpea (Cajanus spp.).

BACKGROUND: Pigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea. RESULTS: A set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme. CONCLUSION: In summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea.

Prenatal BACs-on-Beads™ : a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

OBJECTIVE: Molecular cytogenetic techniques on uncultured prenatal samples are the sole tests applied in some countries in cases with advanced maternal age (AMA) or increased risk after prenatal screening. Moreover, there is a trend to perform invasive prenatal diagnosis (PD) during the first trimester before ultrasound manifestations, so new rapid and reliable assays are necessary to investigate microdeletions not detectable with the conventional karyotype. We report the validation study of the prenatal bacterial artificial chromosomes-on-Beads™ (BoBs™ ; CE-IVD), a bead-based multiplex assay detecting chromosomes 13, 18, 21, X/Y aneuploidies and nine microdeletion regions having an overall detection rate of 1/1700. METHOD: We retrospectively studied 408 selected samples and prospectively tested 212 consecutive samples ascertained for conventional karyotyping. RESULTS: We did not find false-positive results. Triploidies were not detected. Maternal cell contamination of male samples up to 90% was unmasked inspecting gonosome profiles. Mosaic conditions at 20 to 30% were revealed. Failures were due to low amount of DNA. CONCLUSION: Prenatal BoBs™ is a robust technology for the investigation of fetuses with normal karyotype with or without sonographic abnormalities. Running in parallel with the karyotype analysis, it can be proposed instead of rapid FISH or QF-PCR providing rapid results on common aneuploidies and additional information regarding the microdeletion syndromes.

Assessment of structural variation and molecular mapping of insertion sites of Desmar-like elements in the Hessian fly genome.

The Hessian fly (Mayetiola destructor) is an agriculturally important pest of wheat. A mariner element (Desmar1) has been previously identified in the Hessian fly genome. Using Desmar1 as a probe, we isolated individual copies of Desmar-like elements from the Hessian fly genome cloned in bacterial artificial chromosomes (BACs) and studied their structural variability and flanking DNA sequences. The partial Desmar-like copies are relatively more abundant (∼64%) than full length copies (∼36%) in the Hessian fly genome. Most of the full length copies are consistently flanked by an EcoRI restriction site that occurs 32 bp from one end and 66 bp from the other end of the mariner. Using an amplified fragment length polymorphism-PCR (AFLP-PCR) based method, we identified segregating polymorphisms associated with Desmar elements in a F2 mapping population. We were able to use the segregation data to localize the chromosomal position of three Desmar elements by linkage analysis. As paternal chromosomes are eliminated in the Hessian fly during early embryogenesis, two-thirds of the AFLPs were expected to be polymorphic in the mapping population and this was observed for AFLPs anchored to full length Desmar copies but not to the partial copies. Thus, our data indicate that dead and partial Desmar-like copies are probably associated with less polymorphic regions and may represent mariner graveyards in the Hessian fly genome.

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis.

BACKGROUND: The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. RESULTS: Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR) and High Resolution Melt (HRM) analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. CONCLUSIONS: A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.