Asunto(s)
COVID-19/virología , Contaminación de ADN , Mutación , Recombinación Genética/genética , SARS-CoV-2/clasificación , SARS-CoV-2/genética , COVID-19/epidemiología , Chipre/epidemiología , Bases de Datos Genéticas , Humanos , Reproducibilidad de los Resultados , Proyectos de Investigación , Investigadores/normas , SARS-CoV-2/aislamiento & purificación , Análisis de Secuencia/normasRESUMEN
The global spread and continued evolution of SARS-CoV-2 has driven an unprecedented surge in viral genomic surveillance. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine laboratory processes and results. This challenge will increase with the expanding global production of sequences across a variety of laboratories for epidemiological and clinical interpretation, as well as for genomic surveillance of emerging diseases in future outbreaks. We present SDSI + AmpSeq, an approach that uses 96 synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination throughout the sequencing workflow. We apply SDSIs to the ARTIC Consortium's amplicon design, demonstrate their utility and efficiency in a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases and validate them across 6,676 diagnostic samples at multiple laboratories. We establish that SDSI + AmpSeq provides increased confidence in genomic data by detecting and correcting for relatively common, yet previously unobserved modes of error, including spillover and sample swaps, without impacting genome recovery.
Asunto(s)
Cartilla de ADN/normas , SARS-CoV-2/genética , Análisis de Secuencia/normas , COVID-19/diagnóstico , Cartilla de ADN/síntesis química , Genoma Viral/genética , Humanos , Control de Calidad , ARN Viral/genética , Reproducibilidad de los Resultados , Análisis de Secuencia/métodos , Secuenciación Completa del Genoma , Flujo de TrabajoRESUMEN
BACKGROUND: The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. RESULTS: Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach. CONCLUSIONS: The tailed amplicon method we describe represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.