Highly Accurate Chip-Based Resequencing of SARS-CoV-2 Clinical Samples

Hoff, Kendall; Ding, Xun; Carter, Lucas; Duque, John; Lin, Ju Yu; Dung, Samantha; Singh, Priyanka; Sun, Jiayi; Crnogorac, F.; Swaminathan, Radha; Alden, Emily N.; Zhu, Xuechen; Shimada, Ryota; Posavi, Marijan; Hull, Noah; Dinwiddie, Darrell L.; Halász, Ádám; McGall, Glenn H.; Zhou, Wei; Edwards, Jeremy S.

doi:10.1021/acs.langmuir.0c02927

Cited by 13 publications

(20 citation statements)

References 12 publications

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“…For this, we used 5-(3-Aminoallyl)-2′-deoxyuridine-5′-triphosphate (Cy5-dUTP) for labelling of RPA amplicons during the amplification process. A few studies are existing using this technique for labelling PCR products with fluorophores 19 – 21 and other molecules like biotin 21 , 22 , but despite some advantages this is not a commonly used method.…”

Section: Introductionmentioning

confidence: 99%

Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes

Warmt

Fenzel

Henkel

et al. 2021

Sci Rep

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In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the blaCTX-M15 gene in less than 1.6E−03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.

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Section: Introductionmentioning

confidence: 99%

Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes

Warmt

Fenzel

Henkel

et al. 2021

Sci Rep

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“…The tiling array is identical to the DNA chip design described previously 11,13 . In short, each position in the genome (on the sense and antisense strand) is represented on the chip using a group of features composed of 25-mer oligonucleotide probes.…”

Section: Dna Chip Designmentioning

confidence: 99%

“…Samples were prepared as described in our previous work 11 . Briefly, in order to test the robustness and improved sequencing accuracy of the DNA chip by including specific genotyping probes in addition to the tiling array features, we sequenced eight clinical samples from COVID-19 positive patients.…”

Section: Sample Preparation and Hybridizationmentioning

confidence: 99%

“…True variants were separately identified by Illumina short-read sequencing and used to determine the accuracy of the variant calls made by the combined genome tiling array and genotyping probe sets 11 . We then analyzes the accuracy of the results from the chip based sequencing using tiling array features and genotyping probes on four sets of genome positions; all sites in the SARS-CoV-2 genome, the 42 positions of known SARS-CoV-2 variants from the Wyoming clinical samples in the GISAID database as of May 2020, all US SARS-CoV-2 variants from the GISAID database as of May 2020 (US Var, 5055 positions) and confirmed US SARS-CoV-2 variants which have been identified in five or more samples as of May 2020 (USgt5, 1044 positions).…”

Section: Accuracy Analysis Of Samplesmentioning

confidence: 99%

“…In our previous work we introduced a SARS-CoV-2 whole genome resequencing approach utilizing a DNA array chip that can sequence accurately, at single base-pair resolution, the complete SARS-CoV-2 genome 11 . Eight SARS-CoV-2 clinical samples from COVID-19 patients in Wyoming were sequenced and yielded a sequencing accuracy and coverage of 99.86%-99.92% and 98.07%-99.54% respectively.…”

Section: Introductionmentioning

confidence: 99%

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SARS-CoV-2 Variant Identification Using a Genome Tiling Array and Genotyping Probes

Shimada

Alden

Hoff³

et al. 2021

Preprint

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With over three million deaths worldwide attributed to the respiratory disease COVID-19 caused by the novel coronavirus SARS-CoV-2, it is essential that continued efforts be made to track the evolution and spread of the virus globally. We previously presented a rapid and cost-effective method to sequence the entire SARS-CoV-2 genome with 95% coverage and 99.9% accuracy. This method is advantageous for identifying and tracking variants in the SARS-CoV-2 genome when compared to traditional short read sequencing methods which can be time consuming and costly. Herein we present the addition of genotyping probes to our DNA chip which target known SARS-CoV-2 variants. The incorporation of the genotyping probe sets along with the advent of a moving average filter have improved our sequencing coverage and accuracy of the SARS-CoV-2 genome.

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Application of whole‐genome tiling array at Shanghai port, China: An alternative method for SARS‐CoV‐2 surveillance

Liu,

Wang,

Mao

et al. 2023

Journal of Medical Virology

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The ongoing coronavirus disease 2019 (COVID‐19) pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), highlights the critical role of genomic surveillance in tracking rapidly spreading viruses and their evolving lineages. The emergence of the SARS‐CoV‐2 tiling array, a comprehensive tool capable of capturing the entire viral genome, has presented a promising avenue for variants. This study introduces the SARS‐CoV‐2 tiling array as a novel method for port inspection. Using next‐generation sequencing as a benchmark, 35 positive samples underwent sequencing through both methodologies, including the Alpha variant (B.1.1.7), Delta variants (AY.120, AY.122, AY.23.1), and Omicron variants (BA.1, BA.2, BA.2.75, BA.4, BA.5, BE.1, BF.7, BN.1, BQ.1, XBB.1) within the sample set. The whole‐genome tiling array demonstrated successful identification of various sublineages of SARS‐CoV‐2. The average sequencing coverage rates were 99.22% (96.82%−99.92%) for the whole‐genome tiling array and 98.56% (92.81%−99.59%) for Illumina sequencing, respectively. The match rates of these two methods ranged from 92.81%−99.59%, with an average rate of 98.56%. Among the benefits of the whole‐genome tiling array are its cost‐effectiveness and equipment simplification, making it particularly suitable for identifying SARS‐CoV‐2 variants in the front‐line inspection department. The aforementioned findings provide valuable insights into the surveillance of COVID‐19 and present a pragmatic solution for improving quarantine measures at entry points.

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Highly Accurate Chip-Based Resequencing of SARS-CoV-2 Clinical Samples

Cited by 13 publications

References 12 publications

Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes

Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes

SARS-CoV-2 Variant Identification Using a Genome Tiling Array and Genotyping Probes

Application of whole‐genome tiling array at Shanghai port, China: An alternative method for SARS‐CoV‐2 surveillance

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