Next-generation sequencing (NGS) has significantly improved the cost and turnaround time for diagnostic genetic tests. ACMG recommends variant confirmation by an orthogonal method, unless sufficiently high sensitivity and specificity can be demonstrated using NGS alone. Most NGS laboratories make extensive use of Sanger sequencing for secondary confirmation of single nucleotide variants (SNVs) and indels, representing a large fraction of the cost and time required to deliver high quality genetic testing data to clinicians and patients. Despite its established data quality, Sanger is not a high-throughput method by today’s standards from either an assay or analysis standpoint as it can involve…
Targeted sequencing with Sanger as well as short read based high throughput sequencing methods is standard practice in clinical genetic testing. However, many applications beyond SNP detection have remained somewhat obstructed due to technological challenges. With the advent of long reads and high consensus accuracy, SMRT Sequencing overcomes many of the technical hurdles faced by Sanger and NGS approaches, opening a broad range of untapped clinical sequencing opportunities. Flexible multiplexing options, highly adaptable sample preparation method and newly improved two well-developed analysis methods that generate highly-accurate sequencing results, make SMRT Sequencing an adept method for clinical grade targeted sequencing. The…
Ulf Gyllensten speaks about advances in screening for HPV, his predictions for the widespread use of genome sequencing in the clinic, and applications using Single Molecule, Real-Time (SMRT) Sequencing for human genome studies.
Jay Shendure, a Professor in the Department of Genome Sciences at the University of Washington School of Medicine explores the role of exome sequencing in clinical genomics. In this Podcast he discusses his views on the current and future roles of sequencing in diagnosing Mendelian disorders and investigation of complex regions of the genome.
Howard Jacob, Chief Genomics Officer at the HudsonAlpha Institute for Biotechnology, explored the role of genomics in diagnosing rare diseases. In this podcast he shared his views on the economics of clinical sequencing and how long-read sequencing is advancing the ability to sequence an individual’s genome –de novo– and use structural variant calling to make clinical diagnoses. He concluded with the hurdles limiting adoption of clinical sequencing and his vision for the future of genomic medicine.
In this ASHG workshop presentation , Jonas Korlach, CSO of PacBio, walked attendees through recent product updates and the coming technology roadmap. The Sequel System 6.0 release offered major improvements to accuracy, throughput, structural variant calling, and large-insert libraries, he said, showing examples of 35 kb libraries. Looking ahead, Korlach said that the V2 express library preparation product should be available early in 2019, with the new 8M SMRT Cell being introduced sometime later.
To start Day 1 of the PacBio User Group Meeting, Jonas Korlach, PacBio CSO, provides an update on the latest releases and performance metrics for the Sequel II System. The longest reads generated on this system with the SMRT Cell 8M now go beyond 175,000 bases, while maintaining extremely high accuracy. HiFi mode, for example, uses circular consensus sequencing to achieve accuracy of Q40 or even Q50.
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.
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…
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…
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…
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…
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%.
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…
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…