Whole-Genome sequencing and genetic variant analysis of a quarter Horse mare

Abstract Whole-genome sequencing is increasingly being used to investigate the spatial and temporal distribution of viral pathogens including the Severe Acute Respiratory Syndrome Coronavirus Variant 2 (SARS-CoV-2) which is responsible for the ongoing COVID-19 pandemic. In this study, we determined 55 complete genome sequences of SARS-CoV-2 strains isolated from patients from Noakhali, a South-Eastern district in Bangladesh. Variant analysis of our sequenced genomes identified sixteen rare variations in S, six in N, two in M, one in E protein and the S protein variation, Y204F, identified in two of our sequenced strains, has not been reported from any other countries in the GISAID database. Comparison of the prevalence pattern across the country showed GH clade lineages B.1.36 and B.1.36.16 to be abundant in Noakhali and the South-Eastern region of Chittagong when compared to the rest of the country. Phylodynamic analysis of our sequenced genomes revealed that the virus was estimated to be evolving at the rate of 1.065 X 10− 4 subs/site/year. The study results demonstrated the necessity of initiating a concerted, country-wide genomics surveillance effort to determine any novel mutation of functional significance, understanding virus evolution, transmission, and spread in Bangladesh. Short running title: Genome sequencing of Noakhali isolates SARS-Cov-2 in Bangladesh

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SARS-CoV-2 whole-genome sequencing using reverse complement PCR: For easy, fast and accurate outbreak and variant analysis.

Journal of Clinical Virology ◽

10.1016/j.jcv.2021.104993 ◽

2021 ◽

Vol 144 ◽

pp. 104993

Author(s):

Jordy P.M. Coolen ◽

Femke Wolters ◽

Alma Tostmann ◽

Lenneke F.J. van Groningen ◽

Chantal P. Bleeker-Rovers ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Reverse Complement ◽

Variant Analysis

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Multiplex Fragment analysis detects all COVID-19 variants of concern

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.294 ◽

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.

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Impact of DNA source on genetic variant detection from human whole-genome sequencing data

Journal of Medical Genetics ◽

10.1136/jmedgenet-2019-106281 ◽

2019 ◽

Vol 56 (12) ◽

pp. 809-817 ◽

Cited By ~ 7

Author(s):

Brett Trost ◽

Susan Walker ◽

Syed A Haider ◽

Wilson W L Sung ◽

Sergio Pereira ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Variant ◽

Read Depth ◽

Detection Accuracy ◽

Whole Genome ◽

Sequencing Data ◽

Eukaryotic Dna ◽

Variant Detection ◽

Cnv Detection

BackgroundWhole blood is currently the most common DNA source for whole-genome sequencing (WGS), but for studies requiring non-invasive collection, self-collection, greater sample stability or additional tissue references, saliva or buccal samples may be preferred. However, the relative quality of sequencing data and accuracy of genetic variant detection from blood-derived, saliva-derived and buccal-derived DNA need to be thoroughly investigated.MethodsMatched blood, saliva and buccal samples from four unrelated individuals were used to compare sequencing metrics and variant-detection accuracy among these DNA sources.ResultsWe observed significant differences among DNA sources for sequencing quality metrics such as percentage of reads aligned and mean read depth (p<0.05). Differences were negligible in the accuracy of detecting short insertions and deletions; however, the false positive rate for single nucleotide variation detection was slightly higher in some saliva and buccal samples. The sensitivity of copy number variant (CNV) detection was up to 25% higher in blood samples, depending on CNV size and type, and appeared to be worse in saliva and buccal samples with high bacterial concentration. We also show that methylation-based enrichment for eukaryotic DNA in saliva and buccal samples increased alignment rates but also reduced read-depth uniformity, hampering CNV detection.ConclusionFor WGS, we recommend using DNA extracted from blood rather than saliva or buccal swabs; if saliva or buccal samples are used, we recommend against using methylation-based eukaryotic DNA enrichment. All data used in this study are available for further open-science investigation.

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