Analysis Of Complex Genomes Research Articles

Diagnosis of SLC25A46-related pontocerebellar hypoplasia in two siblings with fulminant neonatal course: role of postmortem CT and whole genomic analysis: a case report

BackgroundPontocerebellar hypoplasia (PCH) is increasingly known as a degenerative disease rather than simple “hypoplasia”. At least 21 disease-causing genes have been identified for PCH so far. Because PCH is very heterogenous, prognostic prediction based solely on clinical or radiologic findings is not feasible.Case presentationHere, we report two siblings who had a fulminant neonatal course. The documentation of pontocerebellar hypoplasia by postmortem brain CT imaging in one of the siblings and a subsequent complex and comprehensive whole genome analysis established that both siblings had bi-allelic compound heterozygous variants (a splicing variant and a deletion) in the SLC25A46 gene which encodes a solute carrier protein essential for mitochondrial function. Long-read whole genome sequencing was required to confirm the presence of the deletion. The fulminant courses suggest that SLC25A46-related PCH is an acutely progressive degenerative condition starting in utero, rather than a simple static hypoplasia.ConclusionThe genomic analysis was instrumental and essential to solving the enigma of the unexplained neonatal deaths of these two siblings and to provide accurate genetic counseling.

DECA: scalable XHMM exome copy-number variant calling with ADAM and Apache Spark

BackgroundXHMM is a widely used tool for copy-number variant (CNV) discovery from whole exome sequencing data but can require hours to days to run for large cohorts. A more scalable implementation would reduce the need for specialized computational resources and enable increased exploration of the configuration parameter space to obtain the best possible results.ResultsDECA is a horizontally scalable implementation of the XHMM algorithm using the ADAM framework and Apache Spark that incorporates novel algorithmic optimizations to eliminate unneeded computation. DECA parallelizes XHMM on both multi-core shared memory computers and large shared-nothing Spark clusters. We performed CNV discovery from the read-depth matrix in 2535 exomes in 9.3 min on a 16-core workstation (35.3× speedup vs. XHMM), 12.7 min using 10 executor cores on a Spark cluster (18.8× speedup vs. XHMM), and 9.8 min using 32 executor cores on Amazon AWS’ Elastic MapReduce. We performed CNV discovery from the original BAM files in 292 min using 640 executor cores on a Spark cluster.ConclusionsWe describe DECA’s performance, our algorithmic and implementation enhancements to XHMM to obtain that performance, and our lessons learned porting a complex genome analysis application to ADAM and Spark. ADAM and Apache Spark are a performant and productive platform for implementing large-scale genome analyses, but efficiently utilizing large clusters can require algorithmic optimizations and careful attention to Spark’s configuration parameters.

Electrostatic confinement and manipulation of DNA molecules for genome analysis

Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte "issues" as exploitable advantages by our invention and characterization of the "molecular gate," which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an "electrostatic bottle." This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasmaflorum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

Machine learning models in error and variant detection in high-variation high-throughput sequencing datasets

In high-variation genomics datasets, such as found in metagenomics or complex polyploid genome analysis, error detection and variant calling are impeded by the difficulty in discerning sequencing errors from actual biological variation. Confirming base candidates with high frequency of occurrence is no longer a reliable measure, because of the natural variation and the presence of rare bases.This work employs machine learning models to classify bases into erroneous and rare variations, after preselecting potential error candidates with a weighted frequency measure, which aims to focus on unexpected variations by using the inter-sequence pairwise similarity. Different similarity measures are used to account for different types of datasets. Four machine learning models are tested.

Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat.

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence insitu hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC-labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat-Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.

Development of a D genome specific marker resource for diploid and hexaploid wheat.

BackgroundMapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome.ResultsIn this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing ~ 3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci.ConclusionsIn bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website (http://probes.pw.usda.gov/ATRJM/) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1852-2) contains supplementary material, which is available to authorized users.

RepARK—de novo creation of repeat libraries from whole-genome NGS reads

Generation of repeat libraries is a critical step for analysis of complex genomes. In the era of next-generation sequencing (NGS), such libraries are usually produced using a whole-genome shotgun (WGS) derived reference sequence whose completeness greatly influences the quality of derived repeat libraries. We describe here a de novo repeat assembly method—RepARK (Repetitive motif detection by Assembly of Repetitive K-mers)—which avoids potential biases by using abundant k-mers of NGS WGS reads without requiring a reference genome. For validation, repeat consensuses derived from simulated and real Drosophila melanogaster NGS WGS reads were compared to repeat libraries generated by four established methods. RepARK is orders of magnitude faster than the other methods and generates libraries that are: (i) composed almost entirely of repetitive motifs, (ii) more comprehensive and (iii) almost completely annotated by TEclass. Additionally, we show that the RepARK method is applicable to complex genomes like human and can even serve as a diagnostic tool to identify repetitive sequences contaminating NGS datasets.

Evaluation of SNP Data from the Malus Infinium Array Identifies Challenges for Genetic Analysis of Complex Genomes of Polyploid Origin

High throughput arrays for the simultaneous genotyping of thousands of single-nucleotide polymorphisms (SNPs) have made the rapid genetic characterisation of plant genomes and the development of saturated linkage maps a realistic prospect for many plant species of agronomic importance. However, the correct calling of SNP genotypes in divergent polyploid genomes using array technology can be problematic due to paralogy, and to divergence in probe sequences causing changes in probe binding efficiencies. An Illumina Infinium II whole-genome genotyping array was recently developed for the cultivated apple and used to develop a molecular linkage map for an apple rootstock progeny (M432), but a large proportion of segregating SNPs were not mapped in the progeny, due to unexpected genotype clustering patterns. To investigate the causes of this unexpected clustering we performed BLAST analysis of all probe sequences against the ‘Golden Delicious’ genome sequence and discovered evidence for paralogous annealing sites and probe sequence divergence for a high proportion of probes contained on the array. Following visual re-evaluation of the genotyping data generated for 8,788 SNPs for the M432 progeny using the array, we manually re-scored genotypes at 818 loci and mapped a further 797 markers to the M432 linkage map. The newly mapped markers included the majority of those that could not be mapped previously, as well as loci that were previously scored as monomorphic, but which segregated due to divergence leading to heterozygosity in probe annealing sites. An evaluation of the 8,788 probes in a diverse collection of Malus germplasm showed that more than half the probes returned genotype clustering patterns that were difficult or impossible to interpret reliably, highlighting implications for the use of the array in genome-wide association studies.

HGV2012: Leveraging Next-Generation Technology and Large Datasets to Advance Disease Research

The 13th International Meeting on Human Genome Variation and Complex Genome Analysis (HGV2012: Shanghai, China, 6th-8th September 2012) was a stimulating workshop where researchers from academia and industry explored the latest progress, challenges, and opportunities in genome variation research. Key themes included advancements in next-generation sequencing (NGS) technology, investigation of common and rare diseases, employing NGS in the clinic, utilizing large datasets that leverage biobanks and population-specific cohorts, and exploration of genomic features.

BIOBOARD

INDONESIA – Biomed Analysis: Indonesia needs a central science fund. KOREA – Agilent Technologies helps City of Daegu and Daegu Gyeongbuk Institute of Science and Technology search for biomarkers for neurological diseases. SINGAPORE – New S$6.5m facility at NTU by SCELSE and Carl Zeiss to boost used water treatment and environmental life sciences engineering research in Singapore. SINGAPORE – Chugai expands in Singapore with opening of second satellite research institute. SINGAPORE – Rising Above the Present into the Next-Generation. SINGAPORE – Austrianova Singapore announces new pancreatic cancer models. SINGAPORE – IBN and Fortis Colorectal Hospital establish tissue bank for colorectal cancer research. SINGAPORE – Electronic medical records progress from an enterprise-centric solution to a customer-specific application, finds Frost & Sullivan. SINGAPORE – Global perspectives on Health IT from Harvard Medical School thought leader, Dr Blackford Middleton. AUSTRALIA – World's first fluorescent flowers. UNITED KINGDOM – AbD Serotec introduces bovine and ovine Dendritic Cell Growth Kits and antibodies. USA – Medicago Successfully Completes the Production of More Than Ten Million Doses of H1N1 VLP Influenza Vaccine in One Month. USA – Research validates BioNano genomics' mapping platform for complex human genome analysis. USA – Nine population strategies to stop short of 9 billion. USA – Virtual science libraries to be launched in Central Asia. USA – Vaginal ring may help reduce HIV infections.

Bread matters: a national initiative to profile the genetic diversity of Australian wheat

The large and complex genome of wheat makes genetic and genomic analysis in this important species both expensive and resource intensive. The application of next-generation sequencing technologies is particularly resource intensive, with at least 17 Gbp of sequence data required to obtain minimal (1×) coverage of the genome. A similar volume of data would represent almost 40× coverage of the rice genome. Progress can be made through the establishment of consortia to produce shared genomic resources. Australian wheat genome researchers, working with Bioplatforms Australia, have collaborated in a national initiative to establish a genetic diversity dataset representing Australian wheat germplasm based on whole genome next-generation sequencing data. Here, we describe the establishment and validation of this resource which can provide a model for broader international initiatives for the analysis of large and complex genomes.

HGV2011: Personalized genomic medicine meets the incidentalome

The 12th International Meeting on Human Genome Variation and Complex Genome Analysis (HGV2011: Berkeley, California, USA, 8th-10th September 2011) was a stimulating workshop where researchers from academia and industry explored the latest progress, challenges, and opportunities in genome variation research. Key themes included progress beyond GWAS, variation in human populations, use of sequence data in medical settings, large-scale sequencing data analysis, and bioinformatics approaches to large datasets.

Reversible DNA Immobilization on the Neutravidin Coated Surfaces

We present a novel approach for single DNA molecule Here we present a novel approach of elongation and reversible immobilization of DNA molecules on neutravidin surfaces by simple control of chemical environments. We demonstrate biocompatibility on the neutravidin surface with restriction enzyme reactions for optical mapping and ligase reactions for tethered DNA. Compared to previous approaches, the use of neutravidin surfaces has unique advantages. First, protein coated surfaces provide biocompatible environments favorable for enzyme reactions. Second, DNA is reversibly immobilized on neutravidin surfaces, which can be beneficial for the development of more complex and integrated genome analysis systems. Finally, neutravidin surfaces are able to tether DNA using biotin labels, which provides a variety of possibilities for the development of advanced genome analysis platforms.

Targeted enrichment of genomic DNA regions for next-generation sequencing

In this review, we discuss the latest targeted enrichment methods and aspects of their utilization along with second-generation sequencing for complex genome analysis. In doing so, we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a powerful tool. We explain how targeted enrichment for next-generation sequencing has made great progress in terms of methodology, ease of use and applicability, but emphasize the remaining challenges such as the lack of even coverage across targeted regions. Costs are also considered versus the alternative of whole-genome sequencing which is becoming ever more affordable. We conclude that targeted enrichment is likely to be the most economical option for many years to come in a range of settings.

HGV2009 meeting: bigger and better studies provide more answers and more questions

The 11th International Meeting on Human Genome Variation and Complex Genome Analysis (HGV2009: Tallinn, Estonia, 11th-13th September 2009) provided a stimulating workshop environment where diverse academics and industry representatives explored the latest progress, challenges, and opportunities in relating genome variation to evolution, technology, health, and disease. Key themes included Genome-Wide Association Studies (GWAS), progress beyond GWAS, sequencing developments, and bioinformatics approaches to large-scale datasets.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics.

Plant flow cytometry—Far beyond the stone age

WHEN using flow cytometry (by that time called Impulscytophotometrie, i.e., impulse cytophotometry in Germany) to quantify nuclear DNA content in faba bean (Vicia faba), the German botanist Friedrich Otto Heller could have hardly imagined that in 1973, he laid the foundation stone of a new scientific discipline, plant flow cytometry (1). In fact, his pioneering work received little attention, with just a dozen of citations on the Web of Science , and only a few of his contemporaries recognized the potential of flow cytometry (FCM) for plant sciences. The turning point was the work of David Galbraith, 10 years later, who came with an ingenious method for rapid isolation of cell nuclei by mechanical homogenization of plant tissues using a razor blade (2). This method replaced lengthy enzymatic treatments, provided the conceptual basis for routine analysis of nuclear DNA content and set the stage for the spread of FCM into plant laboratories (3). However, the dominance of biomedical research in the design of commercially available instruments, coupled with their relatively high cost, as well as a rather low interest of both the scientific and the industrial community slowed down the progress. It was not until the early 1990s that the number of publications on plant FCM started to rise markedly (4). The last decade has witnessed an ever-increasing number of applications in both basic and applied research, as well as in industry and plant breeding (5), making plant FCM a vital and dynamic research discipline with a great potential. Plant material has several unique features not paralleled in animals and humans, which makes its analyses by flow cytometry challenging. First, plant cells have rigid walls and are held together by extra cellular matrix to form complex three-dimensional tissues. It is not a trivial task to produce liquid suspensions of single and regularly shaped cells and subcellular particles such as nuclei, mitochondria, chloroplasts, and chromosomes. Other problems are due to the chemical composition of the cytosol. Plant cells produce a vast array of secondary metabolites that may interfere with a particular assay, for example with the staining of nuclear DNA (6). In addition, autofluorescence of some cellular components like the chloroplasts and cell walls can override the weak fluorescence of stained targets. Concerning the methods and protocols, plant FCM suffers from the fact that the majority of them were adopted from other fields (e.g., biomedical research) and only a few attempts have been made to deal with the peculiarities of plant samples. A rather old-fashioned feature of plant FCM is the predominance of single-parameter analyses. This is clearly a consequence of the enormous success of the method for estimation of nuclear DNA content, which can be done quite reliably with just one fluorescence parameter. The use of FCM for addressing other issues (e.g., particle structure and/or volume based on scatter properties) and for multiparameter analyses in particular (e.g., cell cycle studies) has been scarce. Despite this, FCM contributed significantly to a large number of remarkable and exciting results with farreaching implications. Plant breeding (5) and plant population

SNPs Meet CNVs in Genome-Wide Association Studies: HGV2007 Meeting Report

The Ninth Meeting on Human Genome Variation and Complex Genome Analysis was held in Sitges, Spain, in September 2007. This annual meeting, which originally focused on single nucleotide polymorphisms (SNPs), broadened its scope from 2006 onward to encompass the entire range of genomic variability. Maintaining the relatively small format of 200 delegates, the meeting gathered leading investigators in copy number variation (CNV), SNP association studies, ultrasequencing, population genetics, statistical analysis, and database management, as well as young investigators who initiated careers in these fields. The two-and-a-half-day meeting combined sponsorship by several academic institutions and corporate entities in a venue that facilitated interaction and communication between the participants.

Enhancing genetic mapping of complex genomes through the design of highly-multiplexed SNP arrays: application to the large and unsequenced genomes of white spruce and black spruce

BackgroundTo explore the potential value of high-throughput genotyping assays in the analysis of large and complex genomes, we designed two highly multiplexed Illumina bead arrays using the GoldenGate SNP assay for gene mapping in white spruce (Picea glauca [Moench] Voss) and black spruce (Picea mariana [Mill.] B.S.P.).ResultsEach array included 768 SNPs, identified by resequencing genomic DNA from parents of each mapping population. For white spruce and black spruce, respectively, 69.2% and 77.1% of genotyped SNPs had valid GoldenGate assay scores and segregated in the mapping populations. For each of these successful SNPs, on average, valid genotyping scores were obtained for over 99% of progeny. SNP data were integrated to pre-existing ALFP, ESTP, and SSR markers to construct two individual linkage maps and a composite map for white spruce and black spruce genomes. The white spruce composite map contained 821 markers including 348 gene loci. Also, 835 markers including 328 gene loci were positioned on the black spruce composite map. In total, 215 anchor markers (mostly gene markers) were shared between the two species. Considering lineage divergence at least 10 Myr ago between the two spruces, interspecific comparison of homoeologous linkage groups revealed remarkable synteny and marker colinearity.ConclusionThe design of customized highly multiplexed Illumina SNP arrays appears as an efficient procedure to enhance the mapping of expressed genes and make linkage maps more informative and powerful in such species with poorly known genomes. This genotyping approach will open new avenues for co-localizing candidate genes and QTLs, partial genome sequencing, and comparative mapping across conifers.

Seventh international meeting on single nucleotide polymorphism and complex genome analysis: ‘ever bigger scans and an increasingly variable genome’

In September 2005, the seventh international meeting on single nucleotide polymorphism (SNP) and complex genome analysis was held in Hinckley, near Leicester, UK and the meeting was organised by Anthony Brookes, Stephen Chanock, Ivo Gut, Alec Jeffreys and Pui-Yan Kwok. Similar to prior meetings, the 3-day meeting focused on new trends and methods in the analysis of SNPs and complex human disease. A substantial portion of the meeting was devoted to preliminary analyses of data emerging from the International HapMap Consortium and addressed key issues in patterns of recombination, linkage disequilibrium and population genetics. Of great interest were the sessions that addressed SNP analysis in other species and the emerging field of copy number variation. Overall, there have been a number of recent advances in genomics that promise to accelerate the pace of dissecting the genetic basis of many complex diseases in humans-and perhaps in other species.