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Thursday, May 28, 2020

Webinar: Increasing solve rates for rare and Mendelian diseases with long-read sequencing

Dr. Wenger gives attendees an update on PacBio’s long-read sequencing and variant detection capabilities on the Sequel II System and shares recommendations on how to design your own study using HiFi reads. Then, Dr. Sund from Cincinnati Children’s Hospital Medical Center describes how she has used long-read sequencing to solve rare neurological diseases involving complex structural rearrangements that were previously unsolved with standard methods.

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Tuesday, April 21, 2020

Chryseobacterium mulctrae sp. nov., isolated from raw cow’s milk.

A Gram-stain-negative bacterial strain, designated CA10T, was isolated from bovine raw milk sampled in Anseong, Republic of Korea. Cells were yellow-pigmented, aerobic, non-motile bacilli and grew optimally at 30?°C and pH 7.0 on tryptic soy agar without supplementation of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain CA10T belonged to the genus Chryseobacterium, family Flavobacteriaceae, and was most closely related to Chryseobacterium indoltheticum ATCC 27950T (98.75?% similarity). The average nucleotide identity and digital DNA-DNA hybridization values of strain CA10T were 94.4 and 56.9?%, respectively, relative to Chryseobacterium scophthalmum DSM 16779T, being lower than the cut-off…

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Tuesday, April 21, 2020

Allopseudarcicella aquatilis gen. nov., sp. nov., isolated from freshwater.

A Gram-stain-negative, rod-shaped and red-pigmented strain, HME7025T, was isolated from freshwater sampled in the Republic of Korea. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain HME7025T formed a lineage within the family Cytophagaceae of the phylum Bacteroidetes. Strain HME7025T was closely related to the genera Pseudarcicella, Arcicella and Flectobacillus. The 16S rRNA gene sequence similarity values of strain HME7025T were under 94.5?% to its closest phylogenetic neighbours. The major fatty acids of strain HME7025T were iso-C15?:?0 (41.9?%), summed feature 3 (comprising C16?:?1?7c and/or C16?:?1?6c; 12.2?%) and anteiso-C15?:?0 (10.8?%). The major respiratory quinone was menaquinone-7. The major…

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Tuesday, April 21, 2020

Characterization of Reference Materials for Genetic Testing of CYP2D6 Alleles: A GeT-RM Collaborative Project.

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination…

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Tuesday, April 21, 2020

The complete genome sequence and comparative genome analysis of the multi-drug resistant food-borne pathogen Bacillus cereus.

Bacillus cereus is an opportunistic human pathogen causing food-borne gastrointestinal infections and non-gastrointestinal infections worldwide. The strain B. cereus FORC_013 was isolated from fried eel. Its genome was completely sequenced by PacBio technology, analyzed and compared with other complete genome sequences of Bacillus to elucidate the distinct pathogenic features of the strain isolated in South Korea. Genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding tissue-destructive exoenzymes, and pore-forming toxins. In particular, tissue-destructive (hemolysin BL, nonhaemolytic enterotoxins) and cytolytic proteins (cytolysin) were observed in the genome, which damage the plasma membrane of the epithelial cells of the small intestine…

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Tuesday, April 21, 2020

Targeted Long-Read RNA Sequencing Demonstrates Transcriptional Diversity Driven by Splice-Site Variation in MYBPC3.

To date, clinical sequencing has focused on genomic DNA using targeted panels and exome sequencing. Sequencing of a large hypertrophic cardiomyopathy (HCM) cohort revealed that positive identification of a disease-associated variant was returned in only 32% of patients, with an additional 15% receiving inconclusive results. When genome sequencing fails to reveal causative variants, the transcriptome may provide additional diagnostic clarity. A recent study examining patients with genetically undiagnosed muscle disorders found that RNA sequencing, when used as a complement to exome and whole genome sequencing, had an overall diagnosis rate of 35%.

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Tuesday, April 21, 2020

Whole genome sequencing of NDM-1-producing serotype K1 ST23 hypervirulent Klebsiella pneumoniae in China.

The emergence and spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is causing worldwide concern, whereas NDM-producing hvKP is still rare. Here we report the complete genome sequence characteristics of an NDM-1-producing ST23 type clinical hvKP in PR China.Capsular polysaccharide serotyping was performed by PCR. The complete genome sequence of isolate 3214 was obtained using both the Illumina Hiseq platform and Pacbio RS platform. Multilocus sequence type was identified by submitting the genome sequence to mlst 2.0 and the antimicrobial resistance genes and plasmid replicons were identified using ResFinder and PlasmidFinder, respectively. Transferability of the blaNDM-1-bearing plasmid was determined by conjugation…

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Tuesday, April 21, 2020

Single-Molecule Sequencing: Towards Clinical Applications.

In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that…

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Tuesday, April 21, 2020

A 12-kb structural variation in progressive myoclonic epilepsy was newly identified by long-read whole-genome sequencing.

We report a family with progressive myoclonic epilepsy who underwent whole-exome sequencing but was negative for pathogenic variants. Similar clinical courses of a devastating neurodegenerative phenotype of two affected siblings were highly suggestive of a genetic etiology, which indicates that the survey of genetic variation by whole-exome sequencing was not comprehensive. To investigate the presence of a variant that remained unrecognized by standard genetic testing, PacBio long-read sequencing was performed. Structural variant (SV) detection using low-coverage (6×) whole-genome sequencing called 17,165 SVs (7,216 deletions and 9,949 insertions). Our SV selection narrowed down potential candidates to only five SVs (two deletions…

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Tuesday, April 21, 2020

Analyses of four new Caulobacter Phicbkviruses indicate independent lineages.

Bacteriophages with genomes larger than 200 kbp are considered giant phages, and the giant Phicbkviruses are the most frequently isolated Caulobacter crescentus phages. In this study, we compare six bacteriophage genomes that differ from the genomes of the majority of Phicbkviruses. Four of these genomes are much larger than those of the rest of the Phicbkviruses, with genome sizes that are more than 250 kbp. A comparison of 16 Phicbkvirus genomes identified a ‘core genome’ of 69 genes that is present in all of these Phicbkvirus genomes, as well as shared accessory genes and genes that are unique for each…

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Tuesday, April 21, 2020

A Rigorous Interlaboratory Examination of the Need to Confirm Next-Generation Sequencing-Detected Variants with an Orthogonal Method in Clinical Genetic Testing.

Orthogonal confirmation of next-generation sequencing (NGS)-detected germline variants is standard practice, although published studies have suggested that confirmation of the highest-quality calls may not always be necessary. The key question is how laboratories can establish criteria that consistently identify those NGS calls that require confirmation. Most prior studies addressing this question have had limitations: they have been generally of small scale, omitted statistical justification, and explored limited aspects of underlying data. The rigorous definition of criteria that separate high-accuracy NGS calls from those that may or may not be true remains a crucial issue. We analyzed five reference samples and…

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Tuesday, April 21, 2020

Fudania jinshanensis gen. nov., sp. nov., isolated from faeces of the Tibetan antelope (Pantholops hodgsonii) in China.

Two hitherto unknown bacteria (strains 313T and 352) were recovered from the faeces of Tibetan antelopes on the Tibet-Qinghai Plateau, PR China. Cells were rod-shaped and Gram-stain-positive. The optimal growth conditions were at 37?°C and pH 7. The isolates were closely related to Actinotignum sanguinis (92.6?% 16S rRNA gene sequence similarity), Arcanobacterium haemolyticum (92.5?%), Actinotignum schaalii (92.4?%), Actinobaculum massiliense (92.2?%) and Flaviflexus huanghaiensis (91.6?%). Phylogenetic analyses showed that strains 313T and 352 clustered independently in the vicinity of the genera Actinotignum, Actinobaculum and Flaviflexus, but could not be classified clearly as a member of any of these genera. Phylogenomic analysis…

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Tuesday, April 21, 2020

Genetic and biochemical characterization of FRI-3, a novel variant of the Ambler class A carbapenemase FRI-1.

To characterize a new variant of the FRI class A carbapenemase isolated from an MDR clinical Enterobacter cloacae isolate.A carbapenem-resistant E. cloacae was isolated from a rectal swab from a patient in an ICU in Southern Germany. Various phenotypic tests confirmed production of a putative class A carbapenemase. The new bla gene variant, blaFRI-3, and its genetic environment were characterized by WGS. Biochemical characterization was performed by heterologous expression in Escherichia coli TOP10 and by purification of the enzyme with subsequent determination of its kinetic parameters.PCR and sequencing carried out for different class A carbapenemase genes confirmed the presence of…

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Tuesday, April 21, 2020

Genetic, structural, and functional diversity of low and high-affinity siderophores in strains of nitrogen fixing Azotobacter chroococcum.

To increase iron (Fe) bioavailability in surface soils, microbes secrete siderophores, chelators with widely varying Fe affinities. Strains of the soil bacterium Azotobacter chroococcum (AC), plant-growth promoting rhizobacteria used as agricultural inoculants, require high Fe concentrations for aerobic respiration and nitrogen fixation. Recently, A. chroococcum str. NCIMB 8003 was shown to synthesize three siderophore classes: (1) vibrioferrin, a low-affinity a-hydroxy carboxylate (pFe = 18.4), (2) amphibactins, high-affinity tris-hydroxamates, and (3) crochelin A, a high-affinity siderophore with mixed Fe-chelating groups (pFe = 23.9). The relevance and specific functions of these siderophores in AC strains remain unclear. We analyzed the genome and…

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Tuesday, April 21, 2020

Detecting a long insertion variant in SAMD12 by SMRT sequencing: implications of long-read whole-genome sequencing for repeat expansion diseases.

Long-read sequencing technology is now capable of reading single-molecule DNA with an average read length of more than 10?kb, fully enabling the coverage of large structural variations (SVs). This advantage may pave the way for the detection of unprecedented SVs as well as repeat expansions. Pathogenic SVs of only known genes used to be selectively analyzed based on prior knowledge of target DNA sequence. The unbiased application of long-read whole-genome sequencing (WGS) for the detection of pathogenic SVs has just begun. Here, we apply PacBio SMRT sequencing in a Japanese family with benign adult familial myoclonus epilepsy (BAFME). Our SV…

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