Establishing the involvement of the novel gene AGBL5 in retinitis pigmentosa by whole genome sequencing

While more than 250 genes are known to cause inherited retinal degenerations (IRD), nearly 40–50% of families have the genetic basis for their disease unknown. In this study we sought to identify the underlying cause of IRD in a family by whole genome sequence (WGS) analysis. Clinical characterization including standard ophthalmic examination, fundus photography, visual field testing, electroretinography, and review of medical and family history was performed. WGS was performed on affected and unaffected family members using Illumina HiSeq X10. Sequence reads were aligned to hg19 using BWA-MEM and variant calling was performed with Genome Analysis Toolkit. The called variants were annotated with SnpEff v4.11, PolyPhen v2.2.2, and CADD v1.3. Copy number variations were called using Genome STRiP (svtoolkit 2.00.1611) and SpeedSeq software. Variants were filtered to detect rare potentially deleterious variants segregating with disease. Candidate variants were validated by dideoxy sequencing. Clinical evaluation revealed typical adolescent-onset recessive retinitis pigmentosa (arRP) in affected members. WGS identified about 4 million variants in each individual. Two rare and potentially deleterious compound heterozygous variants p.Arg281Cys and p.Arg487* were identified in the gene ATP/GTP binding protein like 5 ( AGBL5) as likely causal variants. No additional variants in IRD genes that segregated with disease were identified. Mutation analysis confirmed the segregation of these variants with the IRD in the pedigree. Homology models indicated destabilization of AGBL5 due to the p.Arg281Cys change. Our findings establish the involvement of mutations in AGBL5 in RP and validate the WGS variant filtering pipeline we designed.

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PSXIV-24 Real-time, on-site whole genome sequencing with oxford nanopore technologies’ MinION

Journal of Animal Science ◽

10.1093/jas/skab235.470 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 257-258

Author(s):

Hanna Ostrovski ◽

Rodrigo Pelicioni Savegnago ◽

Wen Huang ◽

Cedric Gondro

Keyword(s):

Real Time ◽

Ease Of Use ◽

Whole Genome Sequence ◽

Genomic Sequencing ◽

Whole Genome ◽

Illumina Hiseq ◽

Base Calling ◽

Oxford Nanopore ◽

Oxford Nanopore Technologies

Abstract Most quantitative geneticists are traditionally trained for data analysis in genetic evaluation and genomic prediction, but rarely have extensive knowledge of molecular genetics or experience in experimental labs. Recent products, such as those launched by Oxford Nanopore Technologies (ONT), give those quantitative geneticists a comprehensible and hands-on toolkit to explore DNA sequencing. The ‘MinION’, a small DNA sequencer, is of interest for quantitative geneticists due to both the minimal learning curve and the non-proprietary USB connectivity. This device is small enough to be portable, allowing for potential real-time, on-farm sequencing. The objective of this project is to compare the whole genome sequence (WGS) output of the MinION sequencer to that of the Illumina HiSeq 4000. Blood was collected from a 6-month-old Akaushi calf born on a Michigan State University farm. DNA was extracted from the sample using the QIAamp DNA Blood Kit from Qiagen, and library DNA ligation preparation (SQK-LSK109) from ONT was used. After base-calling with guppy software (provided by ONT), the data were preprocessed and experimental runs with the MinION were compared using quality control. Finally, the data were aligned with guppy software, and was compared to the aligned WGS obtained with Illumina HiSeq. Quality results from each MinION indicate that, despite the low amount of sequence collected in each run (~225,303 reads per run), the quality of bases sequenced was high (Q≥7). The aligned data from the Illumina sequencer provided 40x coverage of the genome, with a total of 739,339,742 reads. Although the amount of data obtained with MinION is much smaller than that of Illumina HiSeq, the high quality of MinION’s data combined with its ease of use give an opportunity of genomic sequencing for users who are either inexperienced or do not have access to large genomic sequencing devices.

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Compound heterozygous variants in IFT140 as a cause of nonsyndromic retinitis pigmentosa

Ophthalmic Genetics ◽

10.1080/13816810.2017.1393827 ◽

2017 ◽

Vol 39 (2) ◽

pp. 286-287

Author(s):

Tisiana Low ◽

Anastassios Kostakis ◽

Meena Balasubramanian

Keyword(s):

Retinitis Pigmentosa ◽

Compound Heterozygous ◽

Compound Heterozygous Variants

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Determination and Structural Analysis of the Whole-Genome Sequence of Fusarium equiseti D25-1

10.21203/rs.2.24663/v1 ◽

2020 ◽

Author(s):

Xueping LI ◽

Jianhong Li ◽

Yonghong Qi ◽

Yonggang Liu ◽

Minquan Li

Keyword(s):

Molecular Mechanisms ◽

Gc Content ◽

Whole Genome Sequence ◽

Effective Control ◽

Sequence Length ◽

Comparative Genomic ◽

Whole Genome ◽

Illumina Hiseq ◽

Fusarium Equiseti ◽

Wide Range

Abstract BackgroundFusarium equiseti is a plant pathogen with a wide range of hosts and diverse effects, including probiotic activity. However, the underlying molecular mechanisms remain unclear, hindering its effective control and utilization. In this study, the Illumina HiSeq 4000 and PacBio platforms were used to sequence and assemble the whole genome of Fusarium equiseti D25-1.ResultsThe assembly included 16 fragments with a GC content of 48.01%, gap number of zero, and size of 40,776,005 bp. There were 40,110 exons and 26,281 introns having a total size of 19,787,286 bp and 2,290,434 bp, respectively. The genome had an average copy number of 333, 71, 69, 31, and 108 for tRNAs, rRNAs, sRNAs, snRNAs, and miRNAs, respectively. The total repetitive sequence length was 1,713,918 bp, accounting for 4.2033% of the genome. In total, 13,134 functional genes were annotated, accounting for 94.97% of the total gene number. Toxin-related genes, including two related to zearalenone and 23 related to trichothecene, were identified. A comparative genomic analysis supported the high quality of the F. equiseti assembly, exhibiting good collinearity with the reference strains, 3,483 species-specific genes, and 1,805 core genes. A gene family analysis revealed more than 2,500 single-copy orthologs. F. equiseti was most closely related to Fusarium pseudograminearum based on a phylogenetic analysis at the whole-genome level.ConclusionsOur comprehensive analysis of the whole genome of F. equiseti provides basic data for studies of gene expression, regulatory and functional mechanisms, evolutionary processes, as well as disease prevention and control.

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Analysis of copy number variations in Holstein-Friesian cow genomes based on whole-genome sequence data

Journal of Dairy Science ◽

10.3168/jds.2016-11987 ◽

2017 ◽

Vol 100 (7) ◽

pp. 5515-5525 ◽

Cited By ~ 5

Author(s):

M. Mielczarek ◽

M. Frąszczak ◽

R. Giannico ◽

G. Minozzi ◽

John L. Williams ◽

...

Keyword(s):

Genome Sequence ◽

Copy Number ◽

Sequence Data ◽

Copy Number Variations ◽

Whole Genome Sequence ◽

Whole Genome ◽

Genome Sequence Data ◽

Holstein Friesian

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Occurrence of Crown Gall Disease on Japanese Spindle (Euonymus japonicas var. Green Rocket) Caused by Agrobacterium rosae in Iran

Plant Disease ◽

10.1094/pdis-03-21-0580-pdn ◽

2021 ◽

Author(s):

Hamzeh Mafakheri ◽

Seied Mohsen Taghavi ◽

Sadegh Zarei ◽

Nemanja Kuzmanovic ◽

Ebrahim Osdaghi

Keyword(s):

Crown Gall ◽

De Novo ◽

Negative Control ◽

Whole Genome Sequence ◽

Reca Gene ◽

Post Inoculation ◽

Whole Genome ◽

Bacterial Strains ◽

Illumina Hiseq ◽

Crown Gall Disease

Crown gall disease caused by diverse Agrobacterium species is one of the main biotic constraints in the ornamental plants industry in Iran (Mafakheri et al. 2017). In August 2019, Japanese spindle (Euonymus japonicus var. Green Rocket) plants showing crown gall symptoms were observed in a commercial greenhouse in Tehran, Iran. Infected plants were characterized by a visible overgrowth on their stems and crown. Bacterial isolation from the gall tissues was performed on nutrient agar (NA) and 1A media as described by Moore et al. (2001). The six resulted bacterial strains (A.E1 to A.E6) were evaluated using PCR primer pair F8360/F8361 amplifying a 453 bp DNA fragment in recA gene and confirmed as Agrobacterium sp. (Shams et al. 2013). Pathogenicity of the strains was evaluated in two independent assays on Japanese spindle plantlets as well as 10-15 day old tomato (Solanum lycopersicum cv. Sunseed 6189) and sunflower (Helianthus annuus cv. Armavirski) plants in greenhouse conditions using the needle prick method as described previously (Mafakheri et al. 2019). The reference strain A. radiobacter ICMP 5856 and sterile distilled water were used as positive and negative controls, respectively. Crown gall symptoms appeared 20-25 days post inoculation on the Japanese spindle plantlets as well as tomato and sunflower plants inoculated with the strains isolated in this study, while the negative control plants remained asymptomatic. Koch’s postulates were accomplished by re-isolating on NA medium and PCR-based identification of the inoculated strains from the symptomatic plants. The representative strain A.E1 was subjected to multilocus sequence analysis (MLSA) using the sequences of four housekeeping genes (i.e. atpD, gyrB, recA, and rpoB) as described previously (Mafakheri et al. 2019). MLSA results revealed that the strain A.E1 is phylogenetically closely related to A. rosae. The sequences were deposited into GenBank under the accession numbers MT007962 to MT007965 for atpD, gyrB, recA, and rpoB, respectively. Further, the strain A.E1 was subjected to whole genome sequencing using Illumina HiSeq X platform. DNA extraction was performed using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany), DNA libraries were obtained with Nextera XT DNA Library Prep Kit (Illumina, USA), and de novo sequence assembly was performed using SPAdes genome assembler. The resulting whole genome sequence was deposited into the GenBank database under the accession number JAFJZW000000000. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were calculated among all the type strains of Agrobacterium species/genomospecies using standard criteria as detailed previously (Osdaghi et al. 2020; Chen et al. 2021). The strain A.E1 had 97% ANI and 72% dDDH values with A. rosae strain NCPPB 1650, suggesting that the bacterial strains isolated from Japanese spindle in Iran belong to A. rosae. This is the first report of A. rosae causing crown gall disease on Japanese spindle in Iran. The new crown gall disease could negatively affect the ornamental shrub production industry in central Iran unless strict sanitary measures are taken into the account in the nurseries in these areas. Further nationwide surveys and samplings are warranted to elucidate the economic impact of the pathogen on ornamental plant industry in the country.

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

BMC Neurology ◽

10.1186/s12883-021-02540-x ◽

2022 ◽

Vol 22 (1) ◽

Author(s):

Mamiko Yamada ◽

Hisato Suzuki ◽

Hiroyuki Adachi ◽

Atsuko Noguchi ◽

Fuyuki Miya ◽

...

Keyword(s):

Genomic Analysis ◽

Postmortem Brain ◽

Compound Heterozygous ◽

Whole Genome ◽

Pontocerebellar Hypoplasia ◽

Whole Genome Analysis ◽

Neonatal Deaths ◽

Radiologic Findings ◽

Long Read ◽

Compound Heterozygous Variants

Abstract Background Pontocerebellar 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 presentation Here, 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. Conclusion The 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.

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In trans variant calling reveals enrichment for compound heterozygous variants in genes involved in neuronal development and growth.

Genetics Research ◽

10.1017/s0016672319000065 ◽

2019 ◽

Vol 101 ◽

Cited By ~ 1

Author(s):

Allison J. Cox ◽

Fillan Grady ◽

Gabriel Velez ◽

Vinit B. Mahajan ◽

Polly J. Ferguson ◽

...

Keyword(s):

Multiple Testing ◽

Variant Calling ◽

Epileptic Encephalopathy ◽

European Ancestry ◽

Compound Heterozygous ◽

Recessive Trait ◽

1000 Genomes Project ◽

1000 Genomes ◽

Project Participants ◽

Compound Heterozygous Variants

Abstract Compound heterozygotes occur when different variants at the same locus on both maternal and paternal chromosomes produce a recessive trait. Here we present the tool VarCount for the quantification of variants at the individual level. We used VarCount to characterize compound heterozygous coding variants in patients with epileptic encephalopathy and in the 1000 Genomes Project participants. The Epi4k data contains variants identified by whole exome sequencing in patients with either Lennox-Gastaut Syndrome (LGS) or infantile spasms (IS), as well as their parents. We queried the Epi4k dataset (264 trios) and the phased 1000 Genomes Project data (2504 participants) for recessive variants. To assess enrichment, transcript counts were compared between the Epi4k and 1000 Genomes Project participants using minor allele frequency (MAF) cutoffs of 0.5 and 1.0%, and including all ancestries or only probands of European ancestry. In the Epi4k participants, we found enrichment for rare, compound heterozygous variants in six genes, including three involved in neuronal growth and development – PRTG (p = 0.00086, 1% MAF, combined ancestries), TNC (p = 0.022, 1% MAF, combined ancestries) and MACF1 (p = 0.0245, 0.5% MAF, EU ancestry). Due to the total number of transcripts considered in these analyses, the enrichment detected was not significant after correction for multiple testing and higher powered or prospective studies are necessary to validate the candidacy of these genes. However, PRTG, TNC and MACF1 are potential novel recessive epilepsy genes and our results highlight that compound heterozygous variants should be considered in sporadic epilepsy.

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Simulation of African and non-African low and high coverage whole genome sequence data to assess variant calling approaches

Briefings in Bioinformatics ◽

10.1093/bib/bbaa366 ◽

2020 ◽

Author(s):

Shatha Alosaimi ◽

Noëlle van Biljon ◽

Denis Awany ◽

Prisca K Thami ◽

Joel Defo ◽

...

Keyword(s):

Genetic Diversity ◽

False Positive ◽

Sequence Data ◽

False Negative ◽

Variant Calling ◽

High Rate ◽

Whole Genome Sequence ◽

Whole Genome ◽

Sequence Coverage ◽

High Coverage

Abstract Current variant calling (VC) approaches have been designed to leverage populations of long-range haplotypes and were benchmarked using populations of European descent, whereas most genetic diversity is found in non-European such as Africa populations. Working with these genetically diverse populations, VC tools may produce false positive and false negative results, which may produce misleading conclusions in prioritization of mutations, clinical relevancy and actionability of genes. The most prominent question is which tool or pipeline has a high rate of sensitivity and precision when analysing African data with either low or high sequence coverage, given the high genetic diversity and heterogeneity of this data. Here, a total of 100 synthetic Whole Genome Sequencing (WGS) samples, mimicking the genetics profile of African and European subjects for different specific coverage levels (high/low), have been generated to assess the performance of nine different VC tools on these contrasting datasets. The performances of these tools were assessed in false positive and false negative call rates by comparing the simulated golden variants to the variants identified by each VC tool. Combining our results on sensitivity and positive predictive value (PPV), VarDict [PPV = 0.999 and Matthews correlation coefficient (MCC) = 0.832] and BCFtools (PPV = 0.999 and MCC = 0.813) perform best when using African population data on high and low coverage data. Overall, current VC tools produce high false positive and false negative rates when analysing African compared with European data. This highlights the need for development of VC approaches with high sensitivity and precision tailored for populations characterized by high genetic variations and low linkage disequilibrium.

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Genome-based characterization of two Colombian clinical Providencia rettgeri isolates co-harboring NDM-1, VIM-2, and other β-lactamases

BMC Microbiology ◽

10.1186/s12866-020-02030-z ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Adriana Piza-Buitrago ◽

Verónica Rincón ◽

John Donato ◽

Sandra Yamile Saavedra ◽

Carolina Duarte ◽

...

Keyword(s):

Resistance Genes ◽

Whole Genome Sequence ◽

New Delhi ◽

Whole Genome ◽

Healthcare Associated Infection ◽

Illumina Hiseq ◽

Providencia Rettgeri ◽

The Usa ◽

Tract Infections ◽

Healthcare Associated

Abstract Background Providencia rettgeri is a nosocomial pathogen associated with urinary tract infections and related to Healthcare-Associated Infection (HAI). In recent years isolates producing New Delhi Metallo-β-lactamase (NDM) and other β-lactamases have been reported that reduce the efficiency of clinical antimicrobial treatments. In this study, we analyzed antibiotic resistance, the presence of resistance genes and the clonal relationship of two P. rettgeri isolates obtained from male patients admitted to the same hospital in Bogotá – Colombia, 2015. Results Antibiotic susceptibility profile evaluated by the Kirby-Bauer method revealed that both isolates were resistant to third-generation carbapenems and cephalosporins. Whole-genome sequencing (Illumina HiSeq) followed by SPAdes assembling, Prokka annotation in combination with an in-house Python program and resistance gene detection by ResFinder identified the same six β-lactamase genes in both isolates: blaNDM-1, blaVIM-2, blaCTX-M-15, blaOXA-10, blaCMY-2 and blaTEM-1. Additionally, various resistance genes associated with antibiotic target alteration (arnA, PmrE, PmrF, LpxA, LpxC, gyrB, folP, murA, rpoB, rpsL, tet34) were found and four efflux pumps (RosAB, EmrD, mdtH and cmlA). The additional resistance to gentamicin in one of the two isolates could be explained by a detected SNP in CpxA (Cys191Arg) which is involved in the stress response of the bacterial envelope. Genome BLAST comparison using CGView, the ANI value (99.99%) and the pangenome (using Roary) phylogenetic tree (same clade, small distance) showed high similarity between the isolates. The rMLST analysis indicated that both isolates were typed as rST-61,696, same as the RB151 isolate previously isolated in Bucaramanga, Colombia, 2013, and the FDAARGOS_330 isolate isolated in the USA, 2015. Conclusions We report the coexistence of the carbapenemase genes blaNDM-1, and blaVIM-2, together with the β-lactamase genes blaCTX-M-15, blaOXA-10, blaCMY-2 and blaTEM-1, in P. rettgeri isolates from two patients in Colombia. Whole-genome sequence analysis indicated a circulation of P. rettgeri rST-61,696 strains in America that needs to be investigated further.

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KoVariome: Korean National Standard Reference Variome database of whole genomes with comprehensive SNV, indel, CNV, and SV analyses

10.1101/187096 ◽

2017 ◽

Author(s):

Jungeun Kim ◽

Jessica A. Weber ◽

Sungwoong Jho ◽

Jinho Jang ◽

JeHoon Jun ◽

...

Keyword(s):

Sequence Data ◽

Copy Number Variations ◽

Genetic Variations ◽

Korean Population ◽

National Standard ◽

Whole Genome Sequence ◽

Whole Genome Sequencing Data ◽

Personal Genome ◽

Whole Genome ◽

Sequencing Data

AbstractHigh-coverage whole-genome sequencing data of a single ethnicity can provide a useful catalogue of population-specific genetic variations. Herein, we report a comprehensive analysis of the Korean population, and present the Korean National Standard Reference Variome (KoVariome). As a part of the Korean Personal Genome Project (KPGP), we constructed the KoVariome database using 5.5 terabases of whole genome sequence data from 50 healthy Korean individuals with an average coverage depth of 31×. In total, KoVariome includes 12.7M single-nucleotide variants (SNVs), 1.7M short insertions and deletions (indels), 4K structural variations (SVs), and 3.6K copy number variations (CNVs). Among them, 2.4M (19%) SNVs and 0.4M (24%) indels were identified as novel. We also discovered selective enrichment of 3.8M SNVs and 0.5M indels in Korean individuals, which were used to filter out 1,271 coding-SNVs not originally removed from the 1,000 Genomes Project data when prioritizing disease-causing variants. CNV analyses revealed gene losses related to bone mineral densities and duplicated genes involved in brain development and fat reduction. Finally, KoVariome health records were used to identify novel disease-causing variants in the Korean population, demonstrating the value of high-quality ethnic variation databases for the accurate interpretation of individual genomes and the precise characterization of genetic variations.

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