Whole-genome sequencing in French Canadians from Quebec

Genome-wide association studies (GWAS) have had a tremendous success in the identification of common DNA sequence variants associated with complex human diseases and traits. However, because of their design, GWAS are largely inappropriate to characterize the role of rare and low-frequency DNA variants on human phenotypic variation. Rarer genetic variation is geographically more restricted, supporting the need for local whole-genome sequencing (WGS) efforts to study these variants in specific populations. Here, we present the first large-scale low-pass WGS of the French-Canadian population. Specifically, we sequenced at ~5.6× coverage the whole genome of 1970 French Canadians recruited by the Montreal Heart Institute Biobank and identified 29 million bi-allelic variants (31 % novel), including 19 million variants with a minor allele frequency (MAF) <0.5 %. Genotypes from the WGS data are highly concordant with genotypes obtained by exome array on the same individuals (99.8 %), even when restricting this analysis to rare variants (MAF <0.5, 99.9 %) or heterozygous sites (98.9 %). To further validate our data set, we showed that we can effectively use it to replicate several genetic associations with myocardial infarction risk and blood lipid levels. Furthermore, we analyze the utility of our WGS data set to generate a French-Canadian-specific imputation reference panel and to infer population structure in the Province of Quebec. Our results illustrate the value of low-pass WGS to study the genetics of human diseases in the founder French-Canadian population.     

Whole-genome sequencing of a laboratory-evolved yeast strain

Background

Two categories of introns are known, a common U2 type and a rare U12 type. These two types of introns are removed by distinct spliceosomes. The phylogenetic distribution of spliceosomal RNAs that are characteristic of the U12 spliceosome, i.e. the U11, U12, U4atac and U6atac RNAs, suggest that U12 spliceosomes were lost in many phylogenetic groups. We have now examined the distribution of U2 and U12 introns in many of these groups.

Results

U2 and U12 introns were predicted by making use of available EST and genomic sequences. The results show that in species or branches where U12 spliceosomal components are missing, also U12 type of introns are lacking. Examples are the choanoflagellate Monosiga brevicollis, Entamoeba histolytica, green algae, diatoms, and the fungal lineage Basidiomycota. Furthermore, whereas U12 splicing does not occur in Caenorhabditis elegans, U12 introns as well as U12 snRNAs are present in Trichinella spiralis, which is deeply branching in the nematode tree. A comparison of homologous genes in T. spiralis and C. elegans revealed different mechanisms whereby U12 introns were lost.

Conclusions

The phylogenetic distribution of U12 introns and spliceosomal RNAs give further support to an early origin of U12 dependent splicing. In addition, this distribution identifies a large number of instances during eukaryotic evolution where such splicing was lost.     

Whole-genome sequencing and assembly with high-throughput, short-read technologies

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology.     

Whole-genome sequencing and variant discovery in C. elegans

Massively parallel sequencing instruments enable rapid and inexpensive DNA sequence data production. Because these instruments are new, their data require characterization with respect to accuracy and utility. To address this, we sequenced a Caernohabditis elegans N2 Bristol strain isolate using the Solexa Sequence Analyzer, and compared the reads to the reference genome to characterize the data and to evaluate coverage and representation. Massively parallel sequencing facilitates strain-to-reference comparison for genome-wide sequence variant discovery. Owing to the short-read-length sequences produced, we developed a revised approach to determine the regions of the genome to which short reads could be uniquely mapped. We then aligned Solexa reads from C. elegans strain CB4858 to the reference, and screened for single-nucleotide polymorphisms (SNPs) and small indels. This study demonstrates the utility of massively parallel short read sequencing for whole genome resequencing and for accurate discovery of genome-wide polymorphisms.     

Whole-genome shotgun sequencing of the sulfur-oxidizing chemoautotroph Tetrathiobacter kashmirensis

The chemolithoautotrophic betaproteobacterium Tetrathiobacter kashmirensis belongs to the family Alcaligenaceae and is phylogenetically closely related to pathogens such as Taylorella and Bordetella species. While a complete inorganic sulfur oxidation gene cluster, soxCDYZAXWB, is present in its genome, pathogenicity islands or genes associated with virulence, disease, cellular invasion, and/or intracellular resistance are completely absent.     

Whole-genome sequencing of eukaryotes: From sequencing of DNA fragments to a genome assembly

Rapid advances in sequencing technologies of second- and even third-generation made the whole genome sequencing a routine procedure. However, the methods for assembling of the obtained sequences and its results require special consideration. Modern assemblers are based on heuristic algorithms, which lead to fragmented genome assembly composed of scaffolds and contigs of different lengths, the order of which along the chromosome and belonging to a particular chromosome often remain unknown. In this regard, the resulting genome sequence can only be considered as a draft assembly. The principal improvement in the quality and reliability of a draft assembly can be achieved by targeted sequencing of the genome elements of different size, e.g., chromosomes, chromosomal regions, and DNA fragments cloned in different vectors, as well as using reference genome, optical mapping, and Hi-C technology. This approach, in addition to simplifying the assembly of the genome draft, will more accurately identify numerical and structural chromosomal variations and abnormalities of the genomes of the studied species. In this review, we discuss the key technologies for the genome sequencing and the de novo assembly, as well as different approaches to improve the quality of existing drafts of genome sequences.     

Whole-genome sequencing of sake yeast Saccharomyces cerevisiae Kyokai no. 7

The term 'sake yeast' is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.     

Detection of minor drug-resistant populations by parallel allele-specific sequencing

We developed a highly sensitive parallel allele-specific sequencing (PASS) assay to simultaneously analyze a large number of viral genomes and detect minor drug-resistant populations at approximately 0.1-0.01% levels. Using this assay on samples from individuals infected with human immunodeficiency viruses (HIV), we successfully detected and quantified minor populations of drug-resistant viruses and performed linkage analysis of multiple-drug resistance mutations. This assay may serve as a useful tool to study drug resistance in HIV and other infectious agents.     

Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.     

Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

ABSTRACT: BACKGROUND: The genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome. RESULTS: We identified approximately 9.7 million single nucleotide variants (SNVs) between the Malaysian cynomolgus and the Indian rhesus macaque genomes. Compared with humans, a smaller nonsynonymous/synonymous SNV ratio in the cynomolgus macaque suggests more effective removal of slightly deleterious mutations. Comparison of two cynomolgus (Malaysian and Vietnamese) and two rhesus (Indian and Chinese) macaque genomes, including previously published macaque genomes, suggests that Indochinese cynomolgus macaques have been more affected by gene introgression from rhesus macaques. We further identified 60 nonsynonymous SNVs that completely differentiated the cynomolgus and rhesus macaque genomes, and that could be important candidate variants for determining species-specific responses to drugs and pathogens. The demographic inference using the genome sequence data revealed that Malaysian cynomolgus macaques have experienced at least three population bottlenecks. CONCLUSIONS: This list of whole-genome SNVs will be useful for many future applications, such as an array-based genotyping system for macaque individuals. High-quality whole-genome sequencing of the cynomolgus macaque genome may aid studies on finding genetic differences that are responsible for phenotypic diversity in macaques and may help control genetic backgrounds among individuals.     

Whole-genome shotgun sequencing of Lactobacillus rhamnosus MTCC 5462, a strain with probiotic potential

Lactobacillus rhamnosus MTCC 5462 was isolated from infant gastrointestinal flora. The strain exhibited an ability to reduce cholesterol and stimulate immunity. The strain has exhibited positive results in alleviating gastrointestinal discomfort and good potential as a probiotic. We sequenced the whole genome of the strain and compared it to the published genome sequence of Lactobacillus rhamnosus GG (ATCC 53103).     

Whole-genome sequencing of a Plasmodium vivax isolate from the China-Myanmar border area

Currently, there is a trend of an increasing number of Plasmodiumvivaxmalaria cases in China that are imported across its Southeast Asiaborder, especially in the China-Myanmar border area (CMB). To date, little is knownabout the genetic diversity of P. vivax in this region. In thispaper, we report the first genome sequencing of a P. vivaxisolate(CMB-1) from a vivax malaria patient in CMB. The sequencing data were aligned onto96.43% of the P. vivax Salvador I reference strain (Sal I) genomewith 7.84-fold coverage as well as onto 98.32% of 14 Sal I chromosomes. Usingthe de novo assembly approach, we generated 8,541 scaffolds andassembled a total of 27.1 Mb of sequence into CMB-1 scaffolds. Furthermore, weidentified all 295 known virgenes, which is the largest subtelomericmultigene family in malaria parasites. These results provide an important foundationfor further research onP. vivax population genetics.     

Cytokines and bacterial infections

During the recent 10–15 years a growing amount of knowledge has been accumulated on the role of cytokines in the pathogenesis and resistance to infections caused by nonviral agents, including a wide range of bacteria. Cytokines can be major mediators of the pathogenic effect in some diseases, and represent important defense mechanisms in others. Detailed knowledge on the role of the growing number of recognised cytokines is important, because it may represent means to combat and to prevent diseases caused by such infections.     

Whole-genome patenting

Gene patenting is now a familiar commercial practice, but there is little awareness that several patents claim ownership of the complete genome sequence of a prokaryote or virus. When these patents are analysed and compared to those for other biological entities, it becomes clear that genome patents seek to exploit the genome as an information base and are part of a broader shift towards intangible intellectual property in genomics.     

Whole-genome sequencing of monozygotic twins discordant for schizophrenia indicates multiple genetic risk factors for schizophrenia

Schizophrenia is a common disorder with a high heritability, but its genetic architecture is still elusive.We implemented whole-genome sequencing(WGS) analysis of 8 families with monozygotic(MZ) twin pairs discordant for schizophrenia to assess potential association of de novo mutations(DNMs) or inherited variants with susceptibility to schizophrenia. Eight non-synonymous DNMs(including one splicing site) were identified and shared by twins, which were either located in previously reported schizophrenia risk genes(p.V24689 I mutation in TTN, p.S2506 T mutation in GCN1L1, IVS3+1G T in DOCK1) or had a benign to damaging effect according to in silico prediction analysis. By searching the inherited rare damaging or loss-of-function(LOF) variants and common susceptible alleles from three classes of schizophrenia candidate genes, we were able to distill genetic alterations in several schizophrenia risk genes, including GAD1, PLXNA2, RELN and FEZ1. Four inherited copy number variations(CNVs; including a large deletion at 16p13.11) implicated for schizophrenia were identified in four families, respectively. Most of families carried both missense DNMs and inherited risk variants, which might suggest that DNMs, inherited rare damaging variants and common risk alleles together conferred to schizophrenia susceptibility. Our results support that schizophrenia is caused by a combination of multiple genetic factors, with each DNM/variant showing a relatively small effect size.     

高通量测序诊断多重真菌与细菌感染所致重症肺炎1例

报道1例高通量测序诊断多重真菌与细菌感染性重症肺炎。患者男,67岁,咳嗽咳痰伴发热1周,胸闷2 d。体检:体温39℃,双肺呼吸音粗,可听及明显湿啰音及喘鸣音,未及胸膜摩擦音。实验室检验示白细胞、C-反应蛋白、降钙素原、血糖、血酮体及D-葡聚糖升高。肺部CT示双肺多发肺炎。痰液高通量测序联合病原学检查提示白念珠菌、曲霉菌、肺炎链球菌等多种真菌与细菌混合感染。诊断:重症肺炎。治疗:予利奈唑胺、替加环素联合伏立康唑抗感染,机械通气、补液、降糖、化痰等对症支持治疗。2周后体温恢复正常、病情好转,23 d后病愈出院。     

Transforming clinical microbiology with bacterial genome sequencing

Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here, we review the current status of clinical microbiology and how it has already begun to be transformed by using next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties, such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. We predict that the application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow.