The development of a highly multiplex long-read sequencing protocol for SARS-CoV-2 genomes based on NS-watermark barcodes

GARCIA LABARI IGNACIO; JOAQUÍN EZPELETA; VICTORIA POSNER; VILLANOVA, GABRIELA V.; SOFIA LAVISTA-LLANOS; BULACIO PILAR; SPETALE FLAVIO EZEQUIEL; MURILLO JAVIER; ANGELONE LAURA; PALLETA A; REMES LENICOV F; AGUSTINA CERRI; BOLATTI ELISA MARIA; CASAL PABLO; SPINELLI SILVANA; GIRI ADRIANA; SILVIA ARRANZ; TAPIA, ELIZABETH

Quilmes

Congreso; XI CAB2C; 2021

Background:Viral genome sequencing allows identifying the evolutionary relationships among viruses, monitoring the validity of diagnostic tests, and investigating potential transmission chains. The objective of this work was the development of a protocol for whole-genome SARS-CoV-2 sequencing with compatible costs, accessibility, and processing times to the demands of the COVID-19 emergency.Results:We build upon the amplicon tiling strategy described previously by Quick J. et al 2017 for the rapidwhole-genome virus sequencing of clinical samples. We looked for an alternative to the establishedSARS-CoV-2 ARTIC (amplicon-tiling) sequencing protocol that could take full advantage of portablesequencing machines of low-capital access cost now available in the emerging market of long-readsequencing technologies. We focused on the SARS-CoV-2 multiplex-PCR 1.5 Kb amplification protocol (2x12-plex reactions), originally designed for the expensive and not portable PacBio sequencing machines, and adapted it for a low-cost and portable MinION alternative. We developed a multiplex sequencing protocol for the parallel sequencing of thousands of genomes instead of the dozens currently reported in the literature. Based on the NS-watermark barcoding approach described previously by Ezpeleta J. et al 2017, we selected barcoding sets of increasing size, 12, 48, and 96, out of a major set of 4096, and modified the multiplex-PCR protocol to allow double-end symmetrical-barcoding of amplicon samples with these rather long barcodes (36 nt). Pools of 12, 48, and 96 samples were sequenced together on the MinION sequencer. After base-calling and trimming of sequencing adapters, reads were individually deconvoluted with an in-house script prepared for calling the NS-watermark decoding software. Even for 96 samples, high coverage rates (> 98% of the genome) and depths (> 30X in each amplicon fragment of 1.5 Kb) were obtained. We uploaded more than 200 genome high-quality sequences to the GISAID database, including the first complete SARS-CoV-2 genome from Santa Fe.Conclusions:Our results validate the multiplex sequencing methodology developed with the NS-watermark barcodes that makes it possible to democratize genomic sequencing for the active surveillance of SARS-CoV-2 and may be extended to other emerging viruses in the future.