1. Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow
- Author
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Fernanda de Mello Malta, Carolina Naomi Izo Nakamura, Deyvid Emanuel Amgarten, Pedro Sebe, Marcela de Souza Basqueira, Maria Soares Nobrega, Murilo Castro Cervato, Pedro Lui Nigro Chazanas, Camila Oliveira dos Santos Alves, João Renato Rebello Pinho, Michel Chieregato Gretschischkin, Felipe Camilo Val, Diego Delgado Colombo de Oliveira, Bruna Mascaro Cordeiro de Azevedo, and Rodrigo de Souza Reis
- Subjects
0301 basic medicine ,Computer science ,Science ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Sample (material) ,Economic shortage ,Real-Time Polymerase Chain Reaction ,computer.software_genre ,Sensitivity and Specificity ,Genome ,Article ,Bottleneck ,Workflow ,03 medical and health sciences ,COVID-19 Testing ,0302 clinical medicine ,Virology ,Humans ,Multiplex ,030212 general & internal medicine ,Multidisciplinary ,SARS-CoV-2 ,Infectious-disease diagnostics ,COVID-19 ,High-Throughput Nucleotide Sequencing ,RNA sequencing ,Genomics ,030104 developmental biology ,Molecular Diagnostic Techniques ,Scalability ,Next-generation sequencing ,Medicine ,Data mining ,Microbial genetics ,computer - 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.
- Published
- 2021
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