scholarly journals Multiplex Fragment analysis detects all COVID-19 variants of concern

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S138-S138
Author(s):  
J A SoRelle ◽  
A Clark ◽  
Z Wang ◽  
J Park
Keyword(s):  
New York ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Fragment Analysis ◽  
Deletion Mutations ◽  
Percent Agreement ◽  
Indian Origin ◽  
Allele Specific ◽  
Variant Analysis

Abstract Introduction/Objective The majority of tracking methods have employed whole genome sequencing, which can be very expensive and time consuming. An alternative method has been to use genotyping of specific mutations to identify variants. However, tracking SARS-CoV-2 variants by targeted methods has been a moving target. Most methods only multiplex four targets per reaction, but we have multiplexed 8 targets in a single tube using fragment analysis. Methods/Case Report Fluorescently labeled primers targeted a combination of insertion/ deletion mutations and single nucleotide mutations. The PCR amplified products, amplicons, were separated by capillary electrophoresis. Primers were designed to detect changes in size indicative of insertion or deletion mutations including: ORF1A:Del3675_3677, S:Del69_70, S:Del144, S:Del157_158, S:Del242_244, ORF8:Del119_120, and ORF8:ins28269-28273. Allele-specific primers were designed to detect both the wild-type and mutated versions of S:N501Y, S:E484K, and S:L452R. Residual nasopharyngeal and nasal specimens testing positive for SARS-CoV-2 by RT-PCR or isothermal amplification (IDnow) methods were selected from May 1- June 24, 2021. Variant analysis was performed by multiplex targeted PCR and whole genome sequencing in parallel on the same specimens to determine positive percent agreement. Results (if a Case Study enter NA) Variant analysis was performed on 250 specimens detecting each of the major variants of concern Alpha (B.1.1.7, U.K. origin, n= 108), Beta (B.1.351, South Africa origin, n=3), Gamma (P.1, Brazil origin, n=12), Delta (B.1.617.2, Indian origin, n=17), and Iota (B.1.526, New York, n=5). Some specimens with low viral load were detected by only PCR (n=18), only WGS (n=41), or neither (n=20). Overall positive percent agreement was 95% (163/171). Conclusion This adjustable method robustly and accurately identifies COVID-19 VOCs utilizing a platform amenable to multiple targets (20-40 targets ranging from 100-500b.p. across four fluorescent channels) using equipment commonly found in routine molecular pathology laboratories. Future directions include adjusting targets to detect new variants.

Whole-genome sequencing and comprehensive variant analysis of a Japanese individual using massively parallel sequencing

2010 ◽  
Vol 42 (11) ◽  
pp. 931-936 ◽  
Author(s):  
Akihiro Fujimoto ◽  
Hidewaki Nakagawa ◽  
Naoya Hosono ◽  
Kaoru Nakano ◽  
Tetsuo Abe ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Whole Genome ◽  
Parallel Sequencing ◽  
Variant Analysis ◽  
Japanese Individual

scholarly journals Comprehensive Whole-Genome Sequencing and Reporting of Drug Resistance Profiles on Clinical Cases of Mycobacterium tuberculosis in New York State

2017 ◽  
Vol 55 (6) ◽  
pp. 1871-1882 ◽  
Author(s):  
Joseph Shea ◽  
Tanya A. Halse ◽  
Pascal Lapierre ◽  
Matthew Shudt ◽  
Donna Kohlerschmidt ◽  
...  
Keyword(s):  
New York ◽  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Predictive Value ◽  
New York State ◽  
Whole Genome ◽  
Content Type ◽  
York State

ABSTRACTWhole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterizeMycobacterium tuberculosisand otherM. tuberculosiscomplex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, paired-end WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptible-predictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.

scholarly journals Low-level rifampin resistance and rpoB mutations in Mycobacterium tuberculosis: An analysis of whole-genome sequencing and drug susceptibility test data in New York

2020 ◽  
Author(s):  
Joseph Shea ◽  
Tanya A. Halse ◽  
Donna Kohlerschmidt ◽  
Pascal Lapierre ◽  
Herns A. Modestil ◽  
...  
Keyword(s):  
New York ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Critical Concentration ◽  
Gene Sequencing ◽  
Drug Susceptibility ◽  
Whole Genome ◽  
Drug Resistant ◽  
Low Level ◽  
Indicator Tube

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multi-drug resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of rpoB mutations which do not confer high levels of RIF resistance as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF minimum inhibitory concentrations (MICs) compared to fully susceptible strains, however remain phenotypically susceptible by mycobacteria growth indicator tube (MGIT) testing and have been associated with poor patient outcomes. Here we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 21/2-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 μg/mL in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT, however were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial-gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.

scholarly journals Allele-Specific Quantification of Structural Variations in Cancer Genomes

2016 ◽  
Author(s):  
Yang Li ◽  
Shiguo Zhou ◽  
David C. Schwartz ◽  
Jian Ma
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Graphical Model ◽  
Genome Structure ◽  
Cancer Genome ◽  
Whole Genome ◽  
Structural Variations ◽  
Cancer Genomes ◽  
Allele Specific

AbstractOne of the hallmarks of cancer genome is aneuploidy, resulting in abnormal copy numbers of alleles. Structural variations (SVs) can further modify the aneuploid cancer genomes into a mixture of rearranged genomic segments with extensive range of somatic copy number alterations (CNAs). Indeed, aneuploid cancer genomes have significantly higher rate of CNAs and SVs. However, although methods have been developed to identify SVs and allele-specific copy number of genome (ASCNG) separately, no existing algorithm can simultaneously analyze SVs and ASCNG. Such integrated approach is particularly important to fully understand the complexity of cancer genomes. Here we introduce a new algorithm called Weaver to provide allele-specific quantification of SVs and CNAs in aneuploid cancer genomes. Weaver uses a probabilistic graphical model by utilizing cancer whole genome sequencing data to simultaneously estimate the digital copy number and inter-connectivity of SVs. Our simulation evaluation, comparison with single-molecule Optical Mapping analysis, and real data applications (including MCF-7, HeLa, and TCGA whole genome sequencing samples) demonstrated that Weaver is highly accurate and can greatly refine the analysis of complex cancer genome structure.

scholarly journals Whole genome sequencing–based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations

2019 ◽  
Vol 24 (4) ◽  
Author(s):  
Estefanía Abascal ◽  
Laura Pérez-Lago ◽  
Miguel Martínez-Lirola ◽  
Álvaro Chiner-Oms ◽  
Marta Herranz ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Host Country ◽  
Whole Genome ◽  
Cross Border ◽  
Recent Transmission ◽  
Allele Specific ◽  
Complex Cluster ◽  
Pcr Targeting ◽  
Border Surveillance

Background The analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective. Aim To propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importations Methods We selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster. Results In five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country. Conclusion We provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.

scholarly journals Characterization of emetic and diarrheal Bacillus cereus strains from a 2016 foodborne outbreak using whole-genome sequencing: addressing the microbiological, epidemiological, and bioinformatic challenges

2018 ◽  
Author(s):  
Laura M. Carroll ◽  
Martin Wiedmann ◽  
Manjari Mukherjee ◽  
David C. Nicholas ◽  
Lisa A. Mingle ◽  
...  
Keyword(s):  
New York ◽  
Bacillus Cereus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Fast Food ◽  
Epidemiological Data ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Foodborne Outbreak

AbstractThe Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) has not been routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group strains were obtained from human cases (n =7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (clade III) and B. cereus sensu stricto (clade IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely-related and formed a well-supported clade relative to publicly-available emetic clade III genomes with an identical sequence type (ST 26). When compared to publicly-available emetic clade III ST 26 B. cereus group genomes, the 30 emetic clade III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly-available genomes by a mean of 301.7 to 528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. Based on both WGS and epidemiological data, we hypothesize that the outbreak was a single source outbreak caused by emetic clade III B. cereus belonging to the B. paranthracis species. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.

scholarly journals Whole genome sequencing and rare variant analysis in essential tremor families

PLoS ONE ◽  
2019 ◽  
Vol 14 (8) ◽  
pp. e0220512 ◽  
Author(s):  
Zagaa Odgerel ◽  
Shilpa Sonti ◽  
Nora Hernandez ◽  
Jemin Park ◽  
Ruth Ottman ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Essential Tremor ◽  
Genome Sequencing ◽  
Rare Variant ◽  
Whole Genome ◽  
Rare Variant Analysis ◽  
Variant Analysis

scholarly journals Intragenic DOK7 deletion detected by whole-genome sequencing in congenital myasthenic syndromes

2017 ◽  
Vol 3 (3) ◽  
pp. e152 ◽  
Author(s):  
Yoshiteru Azuma ◽  
Ana Töpf ◽  
Teresinha Evangelista ◽  
Paulo José Lorenzoni ◽  
Andreas Roos ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Congenital Myasthenic Syndromes ◽  
Specific Pcr ◽  
Number Variation ◽  
Allele Specific ◽  
Exercise Induced ◽  
Allele Specific Pcr ◽  
Myasthenic Syndromes

Objective:To identify the genetic cause in a patient affected by ptosis and exercise-induced muscle weakness and diagnosed with congenital myasthenic syndromes (CMS) using whole-genome sequencing (WGS).Methods:Candidate gene screening and WGS analysis were performed in the case. Allele-specific PCR was subsequently performed to confirm the copy number variation (CNV) that was suspected from the WGS results.Results:In addition to the previously reported frameshift mutation c.1124_1127dup, an intragenic 6,261 bp deletion spanning from the 5′ untranslated region to intron 2 of the DOK7 gene was identified by WGS in the patient with CMS. The heterozygous deletion was suspected based on reduced coverage on WGS and confirmed by allele-specific PCR. The breakpoints had microhomology and an inverted repeat, which may have led to the development of the deletion during DNA replication.Conclusions:We report a CMS case with identification of the breakpoints of the intragenic DOK7 deletion using WGS analysis. This case illustrates that CNVs undetected by Sanger sequencing may be identified by WGS and highlights their relevance in the molecular diagnosis of a treatable neurologic condition such as CMS.

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