Whole-Genome Sequencing Revealed a Late-Maturing Isogenic Rice Koshihikari Integrated with Hd16 Gene Derived from an Ise Shrine Mutant

We identified the key genes controlling the late maturation of the Japonica cultivar Isehikari, which was found at Ise Jingu Shrine and matures 6 days later than Koshihikari. We conducted a genetics-based approach through this study. First, the latest mature plants, which flowered later than Isehikari, were segregated in the F2 and F3 generations of Koshihikari×Isehikari. Next, the linkage relationship of a single late-maturing gene with the SSR markers on the long arm of chromosome 3 was inferred by using late-maturing homozygous F2 segregants. Moreover, genetic analyses of late maturity were conducted through the process of six times of continuous backcross with Koshihikari as a recurrent parent by using the late-maturing homozygous F3 line as a nonrecurrent parent, thus developing a late-maturing isogenic Koshihikari (BC6F2). As a result, we elucidated a single late-maturing gene with incomplete dominance that caused the 14-day maturation delay of Koshihikari. The whole-genome sequencing was conducted on both of Koshihikari and the late-maturing isogenic Koshihikari. Then, the SNP call was conducted as the reference genome of Koshihikari. Finally, a single SNP was identified in the key gene Hd16 of the late-maturing isogenic Koshihikari.

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Genomic Investigation into the Virulome, Pathogenicity, Stress Response Factors, Clonal Lineages, and Phylogenetic Relationship of Escherichia coli Strains Isolated from Meat Sources in Ghana

Genes ◽

10.3390/genes11121504 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1504

Author(s):

Frederick Adzitey ◽

Jonathan Asante ◽

Hezekiel M. Kumalo ◽

Rene B. Khan ◽

Anou M. Somboro ◽

...

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Response Factors ◽

Pathogenic Potential ◽

E Coli ◽

Relationship Of ◽

Sequence Types

Escherichia coli are among the most common foodborne pathogens associated with infections reported from meat sources. This study investigated the virulome, pathogenicity, stress response factors, clonal lineages, and the phylogenomic relationship of E. coli isolated from different meat sources in Ghana using whole-genome sequencing. Isolates were screened from five meat sources (beef, chevon, guinea fowl, local chicken, and mutton) and five areas (Aboabo, Central market, Nyorni, Victory cinema, and Tishegu) based in the Tamale Metropolis, Ghana. Following microbial identification, the E. coli strains were subjected to whole-genome sequencing. Comparative visualisation analyses showed different DNA synteny of the strains. The isolates consisted of diverse sequence types (STs) with the most common being ST155 (n = 3/14). Based Upon Related Sequence Types (eBURST) analyses of the study sequence types identified four similar clones, five single-locus variants, and two satellite clones (more distantly) with global curated E. coli STs. All the isolates possessed at least one restriction-modification (R-M) and CRISPR defence system. Further analysis revealed conserved stress response mechanisms (detoxification, osmotic, oxidative, and periplasmic stress) in the strains. Estimation of pathogenicity predicted a higher average probability score (Pscore ≈ 0.937), supporting their pathogenic potential to humans. Diverse virulence genes that were clonal-specific were identified. Phylogenomic tree analyses coupled with metadata insights depicted the high genetic diversity of the E. coli isolates with no correlation with their meat sources and areas. The findings of this bioinformatic analyses further our understanding of E. coli in meat sources and are broadly relevant to the design of contamination control strategies in meat retail settings in Ghana.

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Identification of Rice Large Grain Gene GW2 by Whole-Genome Sequencing of a Large Grain-Isogenic Line Integrated with Japonica Native Gene and Its Linkage Relationship with the Co-integrated Semidwarf Gene d60 on Chromosome 2

International Journal of Molecular Sciences ◽

10.3390/ijms20215442 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5442

Author(s):

Motonori Tomita ◽

Shiho Yazawa ◽

Yoshimasa Uenishi

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Rice Variety ◽

Kernel Weight ◽

Snp Markers ◽

Recurrent Parent ◽

Whole Genome ◽

Isogenic Line ◽

Chromosome 2 ◽

Single Nucleotide

Genetic analysis of “InochinoIchi,” an exceptionally large grain rice variety, was conducted through five continuous backcrosses with Koshihikari as a recurrent parent using the large grain F3 plant in Koshihikari × Inochinoichi as a nonrecurrent parent. Thorough the F2 and all BCnF2 generations, large, medium, and small grain segregated in a 1:2:1 ratio, indicating that the large grain is controlled by a single allele. Mapping by using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers with small grain homozygous segregants in the F2 of Nipponbare × Inochinoichi, revealed linkage with around 7.7 Mb markers from the distal end of the short arm of chromosome 2. Whole-genome sequencing on a large grain isogenic Koshihikari (BC4F2) using next-generation sequencing (NGS) identified a single nucleotide deletion in GW2 gene, which is located 8.1 Mb from the end of chromosome 2, encoding a RING protein with E3 ubiquitin ligase activity. The GW2-integrated isogenic Koshihikari showed a 34% increase in thousand kernel weight compared to Koshihikari, while retaining a taste score of 80. We further developed a large grain/semi-dwarf isogenic Koshihikari integrated with GW2 and the semidwarfing gene d60, which was found to be localized on chromosome 2. The combined genotype secured high yielding while providing robustness to withstand climate change, which can contribute to the New Green Revolution.

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Evaluating the use of whole-genome sequencing for outbreak investigations in the lack of closely related reference genome

Infection Genetics and Evolution ◽

10.1016/j.meegid.2018.01.014 ◽

2018 ◽

Vol 59 ◽

pp. 1-6 ◽

Cited By ~ 4

Author(s):

Mohamed M.H. Abdelbary ◽

Laurence Senn ◽

Estelle Moulin ◽

Guy Prod'hom ◽

Antony Croxatto ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Reference Genome ◽

Whole Genome ◽

Outbreak Investigations

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An Avirulence Gene Cluster in the Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Identified through Genetic Mapping and Whole-Genome Sequencing of a Sexual Population

mSphere ◽

10.1128/msphere.00128-20 ◽

2020 ◽

Vol 5 (3) ◽

Author(s):

Chongjing Xia ◽

Yu Lei ◽

Meinan Wang ◽

Wanquan Chen ◽

Xianming Chen

Keyword(s):

Whole Genome Sequencing ◽

Gene Cluster ◽

Genome Sequencing ◽

Genetic Map ◽

Reference Genome ◽

Puccinia Striiformis ◽

Whole Genome ◽

High Quality ◽

Sexual Population ◽

Content Type

ABSTRACT Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe (yellow) rust, is an obligate, biotrophic fungus. It was difficult to study the genetics of the pathogen due to the lack of sexual reproduction. The recent discovery of alternate hosts for P. striiformis f. sp. tritici makes it possible to study inheritance and map genes involved in its interaction with plant hosts. To identify avirulence (Avr) genes in P. striiformis f. sp. tritici, we developed a segregating population by selfing isolate 12-368 on barberry (Berberis vulgaris) plants under controlled conditions. The dikaryotic sexual population segregated for avirulent/virulent phenotypes on nine Yr single-gene lines. The parental and progeny isolates were whole-genome sequenced at >30× coverage using Illumina HiSeq PE150 technology. A total of 2,637 high-quality markers were discovered by mapping the whole-genome sequencing (WGS) reads to the reference genome of strain 93-210 and used to construct a genetic map, consisting of 41 linkage groups, spanning 7,715.0 centimorgans (cM) and covering 68 Mb of the reference genome. The recombination rate was estimated to be 1.81 ± 2.32 cM/10 kb. Quantitative trait locus analysis mapped six Avr gene loci to the genetic map, including an Avr cluster harboring four Avr genes, AvYr7, AvYr43, AvYr44, and AvYrExp2. Aligning the genetic map to the reference genome identified Avr candidates and narrowed them to a small genomic region (<200 kb). The discovery of the Avr gene cluster is useful for understanding pathogen evolution, and the identification of candidate genes is an important step toward cloning Avr genes for studying molecular mechanisms of pathogen-host interactions. IMPORTANCE Stripe rust is a destructive disease of wheat worldwide. Growing resistant cultivars is the most effective, easy-to-use, economical, and environmentally friendly strategy for the control of the disease. However, P. striiformis f. sp. tritici can produce new virulent races that may circumvent race-specific resistance. Therefore, understanding the genetic basis of the interactions between wheat genes for resistance and P. striiformis f. sp. tritici genes for avirulence is useful for improving cultivar resistance for more effective control of the disease. This study developed a high-quality map that facilitates genomic and genetic studies of important traits related to pathogen pathogenicity and adaptation to different environments and crop cultivars carrying different resistance genes. The information on avirulence/virulence genes identified in this study can be used for guiding breeding programs to select combinations of genes for developing new cultivars with effective resistance to mitigate this devastating disease.

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Outbreak of invasive wound mucormycosis in a burn unit due to multiple strains of Mucor circinelloides f. circinelloides resolved by whole genome sequencing

10.1101/233049 ◽

2017 ◽

Author(s):

Dea Garcia-Hermoso ◽

Alexis Criscuolo ◽

Soo chan Lee ◽

Matthieu Legrand ◽

Marc Chaouat ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Reference Genome ◽

Mucor Circinelloides ◽

Clinical Isolates ◽

Whole Genome ◽

Direct Transmission ◽

Burn Unit ◽

Environmental Reservoir ◽

Multiple Strains

AbstractMucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole genome sequencing (WGS) analysis.We analyzed M. circinelloides isolates from patients in our burn unit (BU1) together with non-outbreak isolates from burn unit 2 (BU2, Paris area) and from France over a two-year period (2013-2015). For each isolate, WGS and a de novo genome assembly was performed from read data extracted from the aligned contig sequences of the reference genome (1006PhL).A total of 21 isolates were sequenced including 14 isolates from six BU1 patients. Phylogenetic classification showed that the clinical isolates clustered in four highly divergent clades. Clade1 contained at least one of the strains from the six epidemiologically-linked BU1 patients. The clinical isolates seemed specific to each patient. Two patients were infected with more than two strains from different clades suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients shared one strain in BU1, suggesting direct transmission or contamination with the same environmental source.WGS coupled with precise epidemiological data and analysis of several isolates per patients revealed in our study a complex situation with both potential cross-transmission and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.ImportanceInvasive wound mucormycosis (IWM) is a severe infection due to the environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used Whole Genome Sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient-specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission between the patients. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on whole genome sequencing and careful sampling.

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Whole Genome Sequencing Identifies Key Genes in Spinal Schwannoma

Frontiers in Genetics ◽

10.3389/fgene.2020.507816 ◽

2020 ◽

Vol 11 ◽

Author(s):

Xin Gao ◽

Li Zhang ◽

Qi Jia ◽

Liang Tang ◽

Wen Guo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Key Genes ◽

Spinal Schwannoma

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The First Kazakh Whole Genomes: The First Report of NGS Data

Central Asian Journal of Global Health ◽

10.5195/cajgh.2014.146 ◽

2014 ◽

Vol 3 ◽

Author(s):

Ainur Akilzhanova ◽

Ulykbek Kairov ◽

Saule Rakhimova ◽

Askhat Molkenov ◽

Arang Rhie ◽

...

Keyword(s):

Next Generation Sequencing ◽

Pilot Study ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Reference Genome ◽

Whole Genome ◽

Base Pairs ◽

Kazakh Population ◽

Whole Genomes ◽

Generation Sequencing

Introduction: The human genome sequence will underpin human biology and medicine in the next century, providing a single, essential reference to all genetic information. Extraordinary technological advances and decreases in the cost of DNA sequencing have made the possibility of whole genome sequencing (WGS) feasible as a highly accessible test for numerous indications. The international project “Genetic architecture of Kazakh population” is well underway to determine the complete DNA. Next generation sequencing is a powerful tool for genetic analysis, which will enable us to uncover the association of loci at specific sites in the genome associated with disease. The aim of this study was to introduce first data on WGS of 6 Kazakh individuals.Methods: This pilot study is among the first WGS performed on 6 healthy Kazakh individuals, using next generation sequencing platform HiSeq2000, Illumina by manufacturer’s protocols. All generated *.bcl files were simultaneously converted and demultiplexed using bcl2fasta application. Alignment of sequence reads performed using bwa-mem against human b19 reference genome. Sorting, removing of intermediate files, *.bam files assembling, and marking duplicates were performed using PicardTools package. GATK haplotype caller tool was used for variant calling. ClinVar, SNPedia, and Cosmic databases were processed to identify clinical genomic variants in 6 Kazakh whole genomes. Java Runtime Environment and R. Bioconductor packages were installed to perform raw data processing and run program scripts.Results: The sequence alignment and mapping procedures on reference genome hg19 of each 6 healthy Kazakh individual were completed. Between 87,308,581,400 and 107,526,741,301 total base pairs were sequenced with average coverage x29.85. Between 98.85% and 99.58% base pairs were totally mapped and on average 96.07% were properly paired. Het/Hom and Ti/Tv ratios for each whole genome ranged from 1.35 to 1.52 and from 2.07 to 2.08, respectively. We compared and analyzed each genome with on existing clinical databases ClinVar, SNPedia, Cosmic and found from 20 to 25, from 269 to 288, from 7 to 12 SNP records, respectively. The availability of a reference Kazakh genome sequences provides the basis for studying the nature of sequence variation, particularly single nucleotide polymorphisms.Conclusion: The first whole genome sequencing of Kazakhs were performed. In this pilot study, we identified SNPs associated with different conditions. Further studies of WGS on Kazakh population are needed to identify possible unique genetic variants in Kazakhs.

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Contaminating DNA in human saliva alters the detection of variants from whole genome sequencing

Scientific Reports ◽

10.1038/s41598-020-76022-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

C. A. Samson ◽

W. Whitford ◽

R. G. Snell ◽

J. C. Jacobsen ◽

K. Lehnert

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Reference Genome ◽

Bacterial Genome ◽

Human Saliva ◽

Reference Sequence ◽

Whole Genome ◽

Sequence Comparisons ◽

Human Reference Genome ◽

A Genome

Abstract Cells obtained from human saliva are commonly used as an alternative DNA source when blood is difficult or less convenient to collect. Although DNA extracted from saliva is considered to be of comparable quality to that derived from blood, recent studies have shown that non-human contaminating DNA derived from saliva can confound whole genome sequencing results. The most concerning complication is that non-human reads align to the human reference genome using standard methodology, which can critically affect the resulting variant genotypes identified in a genome. We identified clusters of anomalous variants in saliva DNA derived reads which aligned in an atypical manner. These reads had only short regions of identity to the human reference sequence, flanked by soft clipped sequence. Sequence comparisons of atypically aligning reads from eight human saliva-derived samples to RefSeq genomes revealed the majority to be of bacterial origin (63.46%). To partition the non-human reads during the alignment step, a decoy of the most prevalent bacterial genome sequences was designed and utilised. This reduced the number of atypically aligning reads when trialled on the eight saliva-derived samples by 44% and most importantly prevented the associated anomalous genotype calls. Saliva derived DNA is often contaminated by DNA from other species. This can lead to non-human reads aligning to the human reference genome using current alignment best-practices, impacting variant identification. This problem can be diminished by using a bacterial decoy in the alignment process.

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Whole genome sequencing of sporadic Burkitt lymphoma in HIV-infected and uninfected patients.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.8577 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 8577-8577

Author(s):

Deborah Ritter ◽

Kimberly Walker ◽

Myoung Kwon ◽

Premal Lulla ◽

Catherine M. Bollard ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Dna Sequences ◽

Burkitt Lymphoma ◽

Structural Variation ◽

Reference Genome ◽

Statistical Significance ◽

Whole Genome ◽

Average Insert Size ◽

Structural Variants

8577 Background: Burkitt Lymphoma is defined by canonical translocations between MYC and immunoglobulin IgH, IgK or IgL (8:14, 8:2, 8:22, respectively), and is commonly associated with HIV. The identification of HIV from sequenced samples is critical to understanding HIV-associated Burkitt Lymphoma. While recent novel gene mutations (ID3 and TCF3) have been implicated in functional roles, concomitant genomic structural variants and the interaction of HIV with structural variation is less well defined. Methods: We sequenced the whole genomes of 15 patients with 100bp paired-end reads on Illumina Hi-Seq platform, resulting in an average insert size of 278 (+/- 63) and coverage of 60X tumor and 30X normal. We included 7 HIV-negative, and 8 HIV-positive subjects. Sequencing reads were mapped to the reference genome using BWA. Large-scale structural variation was detected by the BreakDancer and Crest programs. Functional annotation was used to prioritize structural variants for validation. Single nucleotide variants and small insertions and deletions were detected by CARNAC, a somatic variation discovery pipeline. The subset of WGS reads that failed to align to the human reference genome were tested for the presence of HIV sequences by comparing the unmapped reads to a database of viral DNA sequences which included the common subtypes of HIV defined by Los Alamos. Reads matching HIV or EBV with an expectation value of <10-4 were analyzed to determine virus coverage and viral integration sites. Results: Canonical MYC-IgH translocations were identified in 9/15 (60%) tumor samples, with 2 additional subjects harboring either a deletion or an inversion near exon1 of MYC; 4 had no MYC rearrangement. MYC translocations occurred equally in both groups. TP53 and SMARC4 point mutations were observed recurrently in the HIV uninfected group but not in the HIV infected patients. Variable levels of HIV DNA sequence were observed in normal tissue of all HIV infected patients. Conclusions: Whole genome sequencing has identified known somatic variants in HIV infected and uninfected patients. Two genes, TP53 and SMARC4, appear to be differentially mutated, but additional samples are needed to achieve statistical significance.

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Kevlar: a mapping-free framework for accurate discovery ofde novovariants

10.1101/549154 ◽

2019 ◽

Author(s):

Daniel S. Standage ◽

C. Titus Brown ◽

Fereydoun Hormozdiari

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Reference Genome ◽

De Novo ◽

Probabilistic Approach ◽

Variant Calling ◽

High Sensitivity ◽

Disease Genes ◽

Whole Genome ◽

Variant Discovery

AbstractMotivationDiscovery of genetic variants by whole genome sequencing has proven a powerful approach to study the etiology of complex genetic disorders. Elucidation of all variants is a necessary step in identifying causative variants and disease genes. In particular, there is an increased interest in detection ofde novovariation and investigation of its role in various disorders. State-of-the-art methods for variant discovery rely on mapping reads from each individual to a reference genome and predicting variants from difference observed between the mapped reads and the reference genome. This process typically results in millions of variant predictions, most of which are inherited and irrelevant to the phenotype of interest. To distinguish between inherited variation and novel variation resulting fromde novogermline mutation, whole-genome sequencing of close relatives (especially parents and siblings) is commonly used. However, standard mapping-based approaches tend to have a high false-discovery rate forde novovariant prediction, which in many cases arises from problems with read mapping. This is a particular challenge in predictingde novoindels and structural variants.ResultsWe have developed a mapping-free method, Kevlar, forde novovariant discovery based on direct comparison of sequence content between related individuals. Kevlar identifies high-abundancek-mers unique to the individual of interest and retrieves the reads containing thesek-mers. These reads are easily partitioned into disjoint sets by sharedk-mer content for subsequent locus-by-locus processing and variant calling. Kevlar also utilizes a novel probabilistic approach to score and rank the variant predictions to identify the most likelyde novovariants. We evaluated Kevlar on simulated and real pedigrees, and demonstrate its ability to detect bothde novoSNVs and indels with high sensitivity and specificity.Availabilityhttps://github.com/kevlar-dev/kevlar

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