Covidseq

Manufactured by Illumina

Sourced in United States

The COVIDSeq is a next-generation sequencing (NGS) solution designed for the detection and analysis of the SARS-CoV-2 virus. It provides a comprehensive genomic view of the virus, enabling researchers and healthcare professionals to monitor viral mutations and variants. The COVIDSeq kit includes reagents and protocols for RNA extraction, library preparation, and sequencing on Illumina's NGS platforms.

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Lab products found in correlation

Veriti 96 well thermal cycler, by Thermo Fisher Scientific (2 mentions) Nextseq 550, by Illumina (2 mentions) 2100 bioanalyzer, by Agilent Technologies (2 mentions) Nextseq 500, by Illumina (2 mentions) Nextseq platform, by Illumina (1 mentions) Nextera xt dna library prep kit, by Illumina (1 mentions) Miseq platform, by Illumina (1 mentions) Panther fusion sars cov 2 assay, by Hologic (1 mentions) Aptima sars cov 2 assay, by Hologic (1 mentions) Nextera xt, by Illumina (1 mentions) Nextseq, by Illumina (1 mentions) Ampure xp bead, by Beckman Coulter (1 mentions) Lunascript rt supermix kit, by New England Biolabs (1 mentions) Sciclone g3 ngsx iq workstation, by PerkinElmer (1 mentions) Novaseq 6000, by Illumina (1 mentions) Dna flex, by Illumina (1 mentions)

14 protocols using covidseq

SARS-CoV-2 Genome Sequencing Protocol

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Sequencing of the viral isolates was done by Illumina COVIDSeq protocol on NextSeq 550 platform as per the manufacturer’s instructions. The quality check of the prepared libraries was performed using DNA high sensitivity assay kit on Bioanalyzer 2100 (Agilent Technologies, United States). The concentration of the libraries was assessed on Qubit (Thermo Fisher Scientific Inc., USA). For amplification and cDNA conversion steps during library preparation, Veriti 96-Well Thermal Cycler (Applied Biosystems).

Gautam P., Paul D., Suroliya V., Garg R., Agarwal R., Das S., Kaur U.S., Pandey A., Bhugra A., Tarai B., Bihari C., Sarin S.K, & Gupta E. (2022). SARS-CoV-2 Lineage Tracking, and Evolving Trends Seen during Three Consecutive Peaks of Infection in Delhi, India: a Clinico-Genomic Study. Microbiology Spectrum, 10(2), e02729-21.

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SARS-CoV-2 Genome Sequencing and Epidemiology

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SARS-CoV-2 whole genome sequencing was performed using the Swift Biosciences v2 or Illumina COVID-Seq amplicon tiling platforms as described previously [17] . To determine descriptive epidemiology of the nucleocapsid D399N mutation, SARS-CoV-2 whole genomes were downloaded from GISAID on April 18, 2021 [18] . Figures were generated with custom R code (github.com/greninger-lab/sars_cov2_antigen_test_n_gene), using ggplot [18] , UpSetR [19] , and PANGO lineages [20] (link).

Bourassa L., Perchetti G.A., Phung Q., Lin M.J., Mills M.G., Roychoudhury P., Harmon K.G., Reed J.C, & Greninger A.L. (2021). A SARS-CoV-2 Nucleocapsid Variant that Affects Antigen Test Performance. Journal of Clinical Virology, 141, 104900.

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SARS-CoV-2 Whole-Genome Sequencing Protocol

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Samples with C_t < 31 were randomly selected across collection dates and sites on a weekly basis for SARS‐CoV‐2 whole‐genome sequencing. As part of the Network for Genomics Surveillance in South Africa, samples were submitted to the KwaZulu‐Natal Research Innovation and Sequencing Platform (KRISP), the National Institute for Communicable Diseases (NICD), or sequencing was performed in‐house. Samples were amplified using the ARCTIC V4 primers¹⁵ and libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina) which were sequenced on the MiSeq platform (Illumina) or COVIDSeq (Illumina) library kits were used with the NextSeq platform (Illumina).¹¹, ¹⁶ Sequence reads were assembled using the Genome Detective SARS‐CoV‐2 online tool (https://www.genomedetective.com/app/typingtool/virus/) by KRISP, the Exatype pipeline (https://sars-cov-2.exatype.com/) by the NICD and the Galaxy SARS‐CoV‐2 pipeline (https://usegalaxy.eu/) for in‐house sequence reads. The consensus sequences were uploaded onto the GISAID (https://www.gisaid.org/) for curation (Table S1). Sequences were then downloaded from GISAID for further analysis using the Nextstrain (https://clades.nextstrain.org) online tool for the construction of the phylogenetic trees and the Pangolin lineage assigner (https://pangolin.cog-uk.io) was used to confirm the lineages.

Subramoney K., Mtileni N., Bharuthram A., Davis A., Kalenga B., Rikhotso M., Maphahlele M., Giandhari J., Naidoo Y., Pillay S., Ramphal U., Ramphal Y., Tegally H., Wilkinson E., Mohale T., Ismail A., Mashishi B., Mbenenge N., de Oliveira T., Makatini Z., Fielding B.C, & Treurnicht F.K. (2022). Identification of SARS‐CoV‐2 Omicron variant using spike gene target failure and genotyping assays, Gauteng, South Africa, 2021. Journal of Medical Virology, 94(8), 3676-3684.

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SARS-CoV-2 Variant Identification Pipeline

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All positive samples with Ct values < 30 were submitted to SARS-CoV-2 variant identification. Genomic libraries were prepared using COVIDseq (Illumina) in an automated SP-960 system (Loccus/MGI). To evaluate the input concentration of the generated libraries, they were quantified using the FluorQuant system with a FluorQuant High Sensitivity Detection Kit (Loccus). Pooled libraries were sequenced using iSeq (Illumina).

Elias M.C., Slavov S.N., Lima A.R., Martins A.J., Barros C.R., Moretti D.B., Araujo E.L., Marqueze E.C., Ribeiro G., Ribeiro G.M., Bernardino J.S., Koser J.R., Clemente L.G., Crispin L.A., Alcantara L.C., Coutinho L.L., Giovanetti M., Silva Q.O., Neto R.M., Haddad R., Kashima S., Viala V.L., Covas D.T, & Sampaio S.C. (2023). A traveling SARS-CoV-2 laboratory as part of a pandemic response among vulnerable Brazilian populations. BMC Public Health, 23, 15.

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Detecting SARS-CoV-2 Variants in Healthcare Workers

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Testing of HCW for SARS-CoV-2 was performed using routine diagnostic procedures in the virology laboratory of the University Hospital of Lyon (Hospices Civils de Lyon, HCL) and included: transcription mediated amplification (TMA) (Aptima SARS-CoV-2 Assay, Hologic, Marlborough, US), loop-mediated isothermal amplification (LAMP) (SARS-CoV-2 ID NOW, Abbott, Sligo, Ireland) and RT-qPCR with different kits (Cobas 6800 SARS-CoV-2 assay, Roche, Basel, Switzerland or Panther Fusion SARS-CoV-2 assay, Hologic). To determine the prevalence of SARS-CoV-2 variants of concern (VOC) in HCW, available positive samples with quantification cycle (Cq) < 28 were sequenced using COVIDSeq (Illumina) as previously described [12 (link)]. Libraries were sequenced to 1 M paired-end reads (2 × 100 bp) and data were analysed using the in-house seqmet bioinformatic pipeline (available at https://github.com/genepii/seqmet). Clades and lineages were determined on samples with genome coverage > 90% using Nextclade and PangoLEARN, respectively.

Guibert N., Trepat K., Pozzetto B., Josset L., Fassier J.B., Allatif O., Saker K., Brengel-Pesce K., Walzer T., Vanhems P, & Trouillet-Assant S. (2023). A third vaccine dose equalises the levels of effectiveness and immunogenicity of heterologous or homologous COVID-19 vaccine regimens, Lyon, France, December 2021 to March 2022. Eurosurveillance, 28(15), 2200746.

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SARS-CoV-2 Whole-Genome Sequencing Protocol

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Whole-genome sequencing was performed on RNA extracted from all qRT-PCR-positive samples using the excess discarded ORAcollect•RNA solution. Sequencing was performed using the Illumina COVIDSeq assay (Illumina, San Diego, CA), and sequences were run on an Illumina NextSeq 500 (24 (link)). Full-length genomes for each amplified sample were then assembled through alignment to the Wuhan-Hu-1 reference sequence (NC_045512.2) (25 ) using Bowtie2 (26 (link)). Nucleotide substitutions, insertions, and deletions were identified with LoFreq (27 (link)) Lineage assignment for each genome was carried out using PANGO v.4.0.6 and PANGO data v.1.9 (28 (link)).

Landaverde L., Turcinovic J., Doucette-Stamm L., Gonzales K., Platt J., Connor J.H, & Klapperich C. (2022). Comparison of BinaxNOW and SARS-CoV-2 qRT-PCR Detection of the Omicron Variant from Matched Anterior Nares Swabs. Microbiology Spectrum, 10(6), e01307-22.

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SARS-CoV-2 Whole-Genome Sequencing Protocols

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Whole-genome sequencing on viral RNA was performed using Nextera XT and CovidSeq protocols (Illumina Inc, San Diego, CA, USA). Briefly, for the in-house protocol (Nextera XT), cDNA synthesis was performed using the LunaScript RT SuperMix Kit (New England Biolabs, Ipswich, MA, USA), followed by SARS-CoV-2 whole-genome amplification with multiplex PCR using ARTIC v3 (nCoV-2019) to generate amplicons with overlaps. Amplicons were purified using AmpureXP beads (Beckman Coulter, High Wycombe, UK) and quantified using the Qubit double-strand DNA (dsDNA) High Sensitivity kit (Life Technologies, Carlsbad, CA, USA). Nextera XT library preparation kits were sequenced on a MiSeq (v2, 500 cycles) (Illumina). The commercial Illumina CovidSeq test was performed using paired-end sequencing on an Illumina NextSeq (v2, 150 cycles) according to the manufacturer's instructions followed by data analyses with Illumina DRAGEN CovidSeq Test Pipeline software. Sequence quality was validated using NextStrain. Sequences were uploaded on GISAID (https://www.gisaid.org; strain names starting with hCoV-19/Belgium/UZA-UA) [2 ].

Mertens J., Coppens J., Loens K., Le Mercier M., Xavier B.B., Lammens C., Vandamme S., Jansens H., Goossens H, & Matheeussen V. (2021). Monitoring the SARS-CoV-2 pandemic: screening algorithm with single nucleotide polymorphism detection for the rapid identification of established and emerging variants. Clinical Microbiology and Infection, 28(1), 124-129.

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Robust SARS-CoV-2 Variant Identification

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A subset of samples was sequenced using an alternative amplicon panel (Illumina COVIDseq) to evaluate the robustness of variant identification among more recent samples. The COVIDseq protocol was implemented on the same liquid handler system (PerkinElmer Sciclone G3 NGSx iQ Workstation) and used to prepare libraries from extracted RNA. COVIDseq libraries were sequenced on an Illumina Novaseq6000 instrument using a 1 × 100 read format with a target of 2 million reads per sample.

Shrestha L., Lin M.J., Xie H., Mills M.G., Mohamed Bakhash S.A., Gaur V.P., Livingston R.J., Castor J., Bruce E.A., Botten J.W., Huang M.L., Jerome K.R., Greninger A.L, & Roychoudhury P. (2022). Clinical Performance Characteristics of the Swift Normalase Amplicon Panel for Sensitive Recovery of Severe Acute Respiratory Syndrome Coronavirus 2 Genomes. The Journal of Molecular Diagnostics : JMD, 24(9), 963-976.

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Sequencing SARS-CoV-2 Variants Using Metagenomics

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SARS-CoV-2 positive specimens were sequenced using metagenomic next-generation sequencing^{40 (link)}, Swift Biosciences SNAP panel⁴¹, and/or Illumina COVIDSeq following the manufacturer’s protocol. Sequencing reads were analyzed and visualized using cutadapt 1.9, bwa version 0.7.17, Picard 2.18.15, VarScan 2.3, Annovar 2018Apr16 version for the Longitudinal Analysis of Viral Alleles (LAVA; available at https://github.com/michellejlin/lava) pipeline with references MZ433225.1 for VOC gamma, NC_045512.2 for VOC delta, and OL965129.1 for VOC omicron⁴². Sequencing reads are available in NCBI BioProject PRJNA803552 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA803552).

Lieber C.M., Cox R.M., Sourimant J., Wolf J.D., Juergens K., Phung Q., Saindane M.T., Smith M.K., Sticher Z.M., Kalykhalov A.A., Natchus M.G., Painter G.R., Sakamoto K., Greninger A.L, & Plemper R.K. (2022). SARS-CoV-2 VOC type and biological sex affect molnupiravir efficacy in severe COVID-19 dwarf hamster model. Nature Communications, 13, 4416.

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CHIKV Genome Sequencing During Outbreak

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During the peak of the DENV outbreak in May 2023, a random screening was conducted on 3744 plasma samples from individuals presenting with febrile syndrome related to arbovirus infections at the Basic Health Units of Ribeirão Preto, located in the Northeast part of São Paulo State, Brazil. CHIKV-RNA was detected using an in-house RT-PCR assay based on the method described by Lanciotti et al., 2006 [5] . Whole genome sequencing of CHIKV was performed utilizing the Illumina COVIDSeq protocol (Illumina), with adaptation employing CHIKV-specific primers [6] (link). The sequencing protocol is accessible at protocols.io under "Pathogen Whole Genome Sequencing -Multiplexed amplicon sequencing" [7] . Sample selection was based on the cycle threshold (Ct) value (<30, mean 26.01 ± 8.15). Subsequently, the Epidemiological Service of the municipality was informed, and patients were subject to clinical monitoring until their symptoms were resolved.

de La Roque D.G.L., Santos E.V., Policastro L.R., da Costa P.N.M., Evaristo M., Yamamoto A.Y., Giomo D.B., Torres P.M.A., Gentil D.C.D., Minto E.C.M., Slavov S.N., Fonseca V., Dos Santos Barros C.R., Martins A.J., Calado R.T., Passos L.M.R., Elias M.C., Sampaio S.C., Giovanetti M., Covas D.T., Alcântara L.C.J, & Kashima S. (2024). Exploring the Chikungunya virus landscape in a dengue-endemic Brazilian area. Journal of infection and public health, 17(7).

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