ATTCT and ATTCC repeat expansions in the ATXN10 gene affect disease penetrance of spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10) is an autosomal-dominant disorder caused by an expanded pentanucleotide repeat in the ATXN10 gene. This repeat expansion, when fully penetrant, has a size of 850-4,500 repeats. It has been shown that the repeat composition can be a modifier of disease, e.g., seizures. Here, we describe a Mexican kindred in which we identified both pure (ATTCT)n and mixed (ATTCT)n-(ATTCC)n expansions in the same family. We used amplification-free targeted sequencing and optical genome mapping to decipher the composition of these repeat expansions. We found a considerable degree of mosaicism of the repeat expansion. This mosaicism was confirmed in skin fibroblasts from individuals with ATXN10 expansions with RNAScope in situ hybridization. All affected family members with the mixed ATXN10 repeat expansion showed typical clinical signs of spinocerebellar ataxia and epilepsy. In contrast, individuals with the pure ATXN10 expansion present with Parkinson's disease or are unaffected, even in individuals more than 20 years older than the average age at onset for SCA10. Our findings suggest that the pure (ATTCT)n expansion is non-pathogenic, while repeat interruptions, e.g., (ATTCC)n, are necessary to cause SCA10. This mechanism has been recently described for several other repeat expansions including SCA31 (BEAN1), SCA37 (DAB1), and three loci for benign adult familial myoclonic epilepsy BAFME (SAMD12, TNRC6A, RAPGEF2). Therefore, long-read sequencing and optical genome mapping of the entire genomic structure of repeat expansions are critical for clinical practice and genetic counseling, as variations in the repeat can affect disease penetrance, symptoms, and disease trajectory.

Robust Detection of Somatic Mosaicism and Repeat Interruptions by Long-Read Targeted Sequencing in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.

The Complete Genome Sequence of Xanthomonas theicola, the Causal Agent of Canker on Tea Plants, Reveals Novel Secretion Systems in Clade-1 Xanthomonads.

Xanthomonas theicola is the causal agent of bacterial canker on tea plants. There is no complete genome sequence available for X. theicola, a close relative of the species X. translucens and X. hyacinthi, thus limiting basic research for this group of pathogens. Here, we release a high-quality complete genome sequence for the X. theicola type strain, CFBP 4691T. Single-molecule real-time sequencing with a mean coverage of 264× revealed two contigs of 4,744,641 bp (chromosome) and 40,955 bp (plasmid) in size. Genome mining revealed the presence of nonribosomal peptide synthases, two CRISPR systems, the Xps type 2 secretion system, and the Hrp type 3 secretion system. Surprisingly, this strain encodes an additional type 2 secretion system and a novel type 3 secretion system with enigmatic function, hitherto undescribed for xanthomonads. Four type 3 effector genes were found on complete or partial transposons, suggesting a role of transposons in effector gene evolution and spread. This genome sequence fills an important gap to better understand the biology and evolution of the early-branching xanthomonads, also known as clade-1 xanthomonads.

High-Quality Genome Resource of Xanthomonas hyacinthi Generated via Long-Read Sequencing.

The bacterial plant pathogen Xanthomonas hyacinthi is the causal agent of yellow disease of Hyacinthus and other ornamental plant genera. There is no available complete genome for X. hyacinthi, limiting basic research for this pathogen. Here, we release a high-quality complete genome sequence for the X. hyacinthi type strain, CFBP 1156. Single-molecule real-time (SMRT) sequencing with a mean coverage of 306× revealed two contigs of 4,918,645 and 44,381 bp in size. This was the first characterized plant-disease-causing species of Xanthomonas and this genome provides a resource to better understand the biology of yellow disease of hyacinth.

Genomic Acquisitions in Emerging Populations of Xanthomonas vasicola pv. vasculorum Infecting Corn in the United States and Argentina.

Xanthomonas vasicola pv. vasculorum is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this pathogen have greatly increased in the past years in South America and in the United States. The rapid spread of this disease in North and South America may be due to more favorable environmental conditions, susceptible hosts and/or genomic changes that favored the spread. To understand whether genetic mechanisms exist behind the recent spread of X. vasicola pv. vasculorum, we used comparative genomics to identify gene acquisitions in X. vasicola pv. vasculorum genomes from the United States and Argentina. We sequenced 41 genomes of X. vasicola pv. vasculorum and the related sorghum-infecting X. vasicola pv. holcicola and performed comparative analyses against all available X. vasicola genomes. Time-measured phylogenetic analyses showed that X. vasicola pv. vasculorum strains from the United States and Argentina are closely related and arose from two introductions to North and South America. Gene content comparisons identified clusters of genes enriched in corn X. vasicola pv. vasculorum that showed evidence of horizontal transfer including one cluster corresponding to a prophage found in all X. vasicola pv. vasculorum strains from the United States and Argentina as well as in X. vasicola pv. holcicola strains. In this work, we explore the genomes of an emerging phytopathogen population as a first step toward identifying genetic changes associated with the emergence. The acquisitions identified may contain virulence determinants or other factors associated with the spread of X. vasicola pv. vasculorum in North and South America and will be the subject of future work.

Complete Genome Sequence of Pandoraea pnomenusa TF-18, a Multidrug-Resistant Organism Isolated from the Rhizosphere of Rice (Oryza sativa L. subsp. japonica).

Pandoraea pnomenusa strain TF-18 was isolated from the roots of rice seedlings on selective medium containing four classes of antibiotics for isolation of Burkholderia pseudomallei Using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing technology, we report here a complete genome of 5,499,432 bases, a GC content of 64.8%, and 4,849 coding sequences.

Genome Resource of Barley Bacterial Blight and Leaf Streak Pathogen Xanthomonas translucens pv. translucens strain UPB886.

Xanthomonas translucens pv. translucens causes bacterial leaf streak and bacterial blight diseases of barley. This pathogen limits barley production globally but remains understudied, with limited genomic resources. To better understand the biology of this X. translucens subgroup, we sequenced the complete genome of the X. translucens pv. translucens strain UPB886.

A Pathovar of Xanthomonas oryzae Infecting Wild Grasses Provides Insight Into the Evolution of Pathogenicity in Rice Agroecosystems.

Xanthomonas oryzae (Xo) are globally important rice pathogens. Virulent lineages from Africa and Asia and less virulent strains from the United States have been well characterized. Xanthomonas campestris pv. leersiae (Xcl), first described in 1957, causes bacterial streak on the perennial grass, Leersia hexandra, and is a close relative of Xo. L. hexandra, a member of the Poaceae, is highly similar to rice phylogenetically, is globally ubiquitous around rice paddies, and is a reservoir of pathogenic Xo. We used long read, single molecule real time (SMRT) genome sequences of five strains of Xcl from Burkina Faso, China, Mali, and Uganda to determine the genetic relatedness of this organism with Xo. Novel transcription activator-like effectors (TALEs) were discovered in all five strains of Xcl. Predicted TALE target sequences were identified in the Leersia perrieri genome and compared to rice susceptibility gene homologs. Pathogenicity screening on L. hexandra and diverse rice cultivars confirmed that Xcl are able to colonize rice and produce weak but not progressive symptoms. Overall, based on average nucleotide identity (ANI), type III (T3) effector repertoires, and disease phenotype, we propose to rename Xcl to X. oryzae pv. leersiae (Xol) and use this parallel system to improve understanding of the evolution of bacterial pathogenicity in rice agroecosystems.

A robust targeted sequencing approach for low input and variable quality DNA from clinical samples.

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification-this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.

Human biosample authentication using the high-throughput, cost-effective SNPtrace(TM) system.

Cell lines are the foundation for much of the fundamental research into the mechanisms underlying normal biologic processes and disease mechanisms. It is estimated that 15%-35% of human cell lines are misidentified or contaminated, resulting in a huge waste of resources and publication of false or misleading data. Here we evaluate a panel of 96 single-nucleotide polymorphism (SNP) assays utilizing Fluidigm microfluidics technology for authentication and sex determination of human cell lines. The SNPtrace Panel was tested on 907 human cell lines. Pairwise comparison of these data show the SNPtrace Panel discriminated among identical, related and unrelated pairs of samples with a high degree of confidence, equivalent to short tandem repeat (STR) profiling. We also compared annotated sex calls with those determined by the SNPtrace Panel, STR and Illumina SNP arrays, revealing a high number of male samples are identified as female due to loss of the Y chromosome. Finally we assessed the sensitivity of the SNPtrace Panel to detect intra-human cross-contamination, resulting in detection of as little as 2% contaminating cell population. In conclusion, this study has generated a database of SNP fingerprints for 907 cell lines used in biomedical research and provides a reliable, fast, and economic alternative to STR profiling which can be applied to any human cell line or tissue sample.

Population differentiation of southern Indian male lineages correlates with agricultural expansions predating the caste system.

Previous studies that pooled Indian populations from a wide variety of geographical locations, have obtained contradictory conclusions about the processes of the establishment of the Varna caste system and its genetic impact on the origins and demographic histories of Indian populations. To further investigate these questions we took advantage that both Y chromosome and caste designation are paternally inherited, and genotyped 1,680 Y chromosomes representing 12 tribal and 19 non-tribal (caste) endogamous populations from the predominantly Dravidian-speaking Tamil Nadu state in the southernmost part of India. Tribes and castes were both characterized by an overwhelming proportion of putatively Indian autochthonous Y-chromosomal haplogroups (H-M69, F-M89, R1a1-M17, L1-M27, R2-M124, and C5-M356; 81% combined) with a shared genetic heritage dating back to the late Pleistocene (10-30 Kya), suggesting that more recent Holocene migrations from western Eurasia contributed <20% of the male lineages. We found strong evidence for genetic structure, associated primarily with the current mode of subsistence. Coalescence analysis suggested that the social stratification was established 4-6 Kya and there was little admixture during the last 3 Kya, implying a minimal genetic impact of the Varna (caste) system from the historically-documented Brahmin migrations into the area. In contrast, the overall Y-chromosomal patterns, the time depth of population diversifications and the period of differentiation were best explained by the emergence of agricultural technology in South Asia. These results highlight the utility of detailed local genetic studies within India, without prior assumptions about the importance of Varna rank status for population grouping, to obtain new insights into the relative influences of past demographic events for the population structure of the whole of modern India.

Multiplex single-nucleotide polymorphism typing of the human Y chromosome using TaqMan probes.

BACKGROUND: The analysis of human Y-chromosome variation in the context of population genetics and forensics requires the genotyping of dozens to hundreds of selected single-nucleotide polymorphisms (SNPs). In the present study, we developed a 121-plex (121 SNPs in a single array) TaqMan array capable of distinguishing most haplogroups and subhaplogroups on the Y-chromosome human phylogeny in Europe. RESULTS: We present data from 264 samples from several European areas and ethnic groups. The array developed in this study shows >99% accuracy of assignation to the Y human phylogeny (with an average call rate of genotypes >96%). CONCLUSIONS: We have created and evaluated a robust and accurate Y-chromosome multiplex which minimises the possible errors due to mixup when typing the same sample in several independent reactions.

A comprehensive linkage map of the dog genome.

We have leveraged the reference sequence of a boxer to construct the first complete linkage map for the domestic dog. The new map improves access to the dog's unique biology, from human disease counterparts to fascinating evolutionary adaptations. The map was constructed with approximately 3000 microsatellite markers developed from the reference sequence. Familial resources afforded 450 mostly phase-known meioses for map assembly. The genotype data supported a framework map with approximately 1500 loci. An additional approximately 1500 markers served as map validators, contributing modestly to estimates of recombination rate but supporting the framework content. Data from approximately 22,000 SNPs informing on a subset of meioses supported map integrity. The sex-averaged map extended 21 M and revealed marked region- and sex-specific differences in recombination rate. The map will enable empiric coverage estimates and multipoint linkage analysis. Knowledge of the variation in recombination rate will also inform on genomewide patterns of linkage disequilibrium (LD), and thus benefit association, selective sweep, and phylogenetic mapping approaches. The computational and wet-bench strategies can be applied to the reference genome of any nonmodel organism to assemble a de novo linkage map.

The prevalence of folate-remedial MTHFR enzyme variants in humans.

Studies of rare, inborn metabolic diseases establish that the phenotypes of some mutations in vitamin-dependent enzymes can be suppressed by supplementation of the cognate vitamin, which restores function of the defective enzyme. To determine whether polymorphisms exist that more subtly affect enzymes yet are augmentable in the same way, we sequenced the coding region of a prototypical vitamin-dependent enzyme, methylenetetrahydrofolate reductase (MTHFR), from 564 individuals of diverse ethnicities. All nonsynonymous changes were evaluated in functional in vivo assays in Saccharomyces cerevisiae to identify enzymatic defects and folate remediability of impaired alleles. We identified 14 nonsynonymous changes: 11 alleles with minor allele frequencies <1% and 3 common alleles (A222V, E429A, and R594Q). Four of 11 low-frequency alleles affected enzyme function, as did A222V. Of the five impaired alleles, four could be restored to normal functionality by elevating intracellular folate levels. All five impaired alleles mapped to the N-terminal catalytic domain of the enzyme, whereas changes in the C-terminal regulatory domain had little effect on activity. Impaired activity correlated with the phosphorylation state of MTHFR, with more severe mutations resulting in lower abundance of the phosphorylated protein. Significantly, diploid yeast heterozygous for mutant alleles were impaired for growth, particularly with lower folate supplementation. These results suggested that multiple less-frequent alleles, in aggregate, might significantly contribute to metabolic dysfunction. Furthermore, vitamin remediation of mutant enzymes may be a common phenomenon in certain domains of proteins.

A second-generation combined linkage physical map of the human genome.

We have completed a second-generation linkage map that incorporates sequence-based positional information. This new map, the Rutgers Map v.2, includes 28,121 polymorphic markers with physical positions corroborated by recombination-based data. Sex-averaged and sex-specific linkage map distances, along with confidence intervals, have been estimated for all map intervals. In addition, a regression-based smoothed map is provided that facilitates interpolation of positions of unmapped markers on this map. With nearly twice as many markers as our first-generation map, the Rutgers Map continues to be a unique and comprehensive resource for obtaining genetic map information for large sets of polymorphic markers.

The linkage disequilibrium maps of three human chromosomes across four populations reflect their demographic history and a common underlying recombination pattern.

The extent and patterns of linkage disequilibrium (LD) determine the feasibility of association studies to map genes that underlie complex traits. Here we present a comparison of the patterns of LD across four major human populations (African-American, Caucasian, Chinese, and Japanese) with a high-resolution single-nucleotide polymorphism (SNP) map covering almost the entire length of chromosomes 6, 21, and 22. We constructed metric LD maps formulated such that the units measure the extent of useful LD for association mapping. LD reaches almost twice as far in chromosome 6 as in chromosomes 21 or 22, in agreement with their differences in recombination rates. By all measures used, out-of-Africa populations showed over a third more LD than African-Americans, highlighting the role of the population's demography in shaping the patterns of LD. Despite those differences, the long-range contour of the LD maps is remarkably similar across the four populations, presumably reflecting common localization of recombination hot spots. Our results have practical implications for the rational design and selection of SNPs for disease association studies.

The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping.

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.

New generation pharmacogenomic tools: a SNP linkage disequilibrium Map, validated SNP assay resource, and high-throughput instrumentation system for large-scale genetic studies.

Since public and private efforts announced the first draft of the human genome last year, researchers have reported great numbers of single nucleotide polymorphisms (SNPs). We believe that the availability of well-mapped, quality SNP markers constitutes the gateway to a revolution in genetics and personalized medicine that will lead to better diagnosis and treatment of common complex disorders. A new generation of tools and public SNP resources for pharmacogenomic and genetic studies--specifically for candidate-gene, candidate-region, and whole-genome association studies--will form part of the new scientific landscape. This will only be possible through the greater accessibility of SNP resources and superior high-throughput instrumentation-assay systems that enable affordable, highly productive large-scale genetic studies. We are contributing to this effort by developing a high-quality linkage disequilibrium SNP marker map and an accompanying set of ready-to-use, validated SNP assays across every gene in the human genome. This effort incorporates both the public sequence and SNP data sources, and Celera Genomics' human genome assembly and enormous resource ofphysically mapped SNPs (approximately 4,000,000 unique records). This article discusses our approach and methodology for designing the map, choosing quality SNPs, designing and validating these assays, and obtaining population frequency ofthe polymorphisms. We also discuss an advanced, high-performance SNP assay chemisty--a new generation of the TaqMan probe-based, 5' nuclease assay-and high-throughput instrumentation-software system for large-scale genotyping. We provide the new SNP map and validation information, validated SNP assays and reagents, and instrumentation systems as a novel resource for genetic discoveries.