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Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow.
de Mello Malta, Fernanda; Amgarten, Deyvid; Val, Felipe Camilo; Cervato, Murilo Castro; de Azevedo, Bruna Mascaro Cordeiro; de Souza Basqueira, Marcela; Dos Santos Alves, Camila Oliveira; Nobrega, Maria Soares; de Souza Reis, Rodrigo; Sebe, Pedro; Gretschischkin, Michel Chieregato; de Oliveira, Diego Delgado Colombo; Nakamura, Carolina Naomi Izo; Chazanas, Pedro Lui Nigro; Pinho, João Renato Rebello.
  • de Mello Malta F; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Amgarten D; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Val FC; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Cervato MC; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • de Azevedo BMC; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • de Souza Basqueira M; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Dos Santos Alves CO; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Nobrega MS; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • de Souza Reis R; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Sebe P; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Gretschischkin MC; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • de Oliveira DDC; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Nakamura CNI; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Chazanas PLN; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Pinho JRR; Laboratorio de Técnicas Especiais, Hospital Israelita Albert Einstein, São Paulo, Brazil. joao.pinho@einstein.br.
Sci Rep ; 11(1): 7122, 2021 03 29.
Article in English | MEDLINE | ID: covidwho-1157917
Preprint
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ABSTRACT
Since the first reported case of the new coronavirus infection in Wuhan, China, researchers and governments have witnessed an unseen rise in the number of cases. Thanks to the rapid work of Chinese scientists, the pathogen now called SARS-CoV-2 has been identified and its whole genome was deposited in public databases by early January 2020. The availability of the genome has allowed researchers to develop Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays, which are now the gold-standard for molecular diagnosis of the respiratory syndrome COVID19. Because of the rising number of cases and rapid spreading, the world has been facing a shortage of RT-PCR supplies, especially the ones involved in RNA extraction. This has been a major bottleneck to increase testing capacity in many countries that do not significantly manufacture these supplies, such as Brazil. Additionally, RT-qPCR scalability is highly dependent on equipment that usually performs testing of 96 samples at a time. In this work, we describe a cost-effective molecular NGS-based test for diagnosis of COVID19, which uses a single-step RNA extraction and presents high scalability and accuracy when compared to the gold-standard RT-qPCR. A single run of the NGS-based test using the Illumina NextSeq 550 mid-end sequencing equipment is able to multiplex 1,536 patient's samples, providing individual semi-qualitative results (detected, not detected). Detected results are provided with fragments per million (FPM) values, which was demonstrated to correlate with RT-qPCR Cycle Threshold (CT) values. Besides, usage of the high-end Illumina Novaseq platform may yield diagnostic for up to 6144 samples in a single run. Performance results when compared with RT-qPCR show general accuracy of 96%, and 98% when only samples with CT values (gene N) lower than 30 are considered. We have also developed an online platform, termed VarsVID, to help test executors to easily scale testing numbers. Sample registering, wet-lab worksheets generation, sample sheet for sequencing and results' display are all features provided by VarsVID. Altogether, these results will contribute to control COVID19 pandemics.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Molecular Diagnostic Techniques / High-Throughput Nucleotide Sequencing / COVID-19 Testing / COVID-19 Type of study: Diagnostic study / Observational study / Qualitative research Limits: Humans Language: English Journal: Sci Rep Year: 2021 Document Type: Article Affiliation country: S41598-021-86498-3

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Molecular Diagnostic Techniques / High-Throughput Nucleotide Sequencing / COVID-19 Testing / COVID-19 Type of study: Diagnostic study / Observational study / Qualitative research Limits: Humans Language: English Journal: Sci Rep Year: 2021 Document Type: Article Affiliation country: S41598-021-86498-3