MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples

Abstract

32The novel coronavirus disease 2019 pandemic poses a serious public health 33 risk. Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-34 CoV-2) from clinical samples is crucial for the understanding of viral spread and viral 35 evolution, as well as for vaccine development. Existing sample preparation methods for 36 viral genome sequencing are demanding on user technique and time, and thus not ideal 37 for time-sensitive clinical samples; these methods are also not optimized for high 38 performance on viral genomes. We have developed MetagenomIc RNA EnRichment 39 VirAl sequencing (MINERVA), a facile, practical, and robust approach for metagenomic 40 and deep viral sequencing from clinical samples. This approach uses direct tagmentation 41 of RNA/DNA hybrids using Tn5 transposase to greatly simplify the sequencing library 42 construction process, while subsequent targeted enrichment can generate viral genomes 43 with high sensitivity, coverage, and depth. We demonstrate the utility of MINERVA on 44 pharyngeal, sputum and stool samples collected from COVID-19 patients, successfully 45 obtaining both whole metatranscriptomes and complete high-depth high-coverage SARS-46 CoV-2 genomes from these clinical samples, with high yield and robustness. MINERVA 47 is compatible with clinical nucleic extracts containing carrier RNA. With a shortened 48 hands-on time from sample to virus-enriched sequencing-ready library, this rapid, 49 versatile, and clinic-friendly approach will facilitate monitoring of viral genetic variations 50 during outbreaks, both current and future. 51 52 53 Introduction 54As of April 24, 2020, the ongoing COVID-19 viral pandemic has affected more than 2.6 55 million people in over 200 countries and territories around the world, and has claimed 56 more than 180 thousand lives. Closely monitoring the genetic diversity and distribution of 57 viral strains at the population level is essential for epidemiological tracking, and for 58 understanding viral evolution and transmission; additionally examining the viral 59 heterogeneity within a single individual is imperative for diagnosis and treatment. The 60 disease-causing pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-61 CoV-2), was identified from early disease cases and its draft genome sequenced within 62 weeks, thanks to the rapid responses from researchers around the world(1, 2). The initial 63 SARS-CoV-2 draft genome was obtained independently from the same early COVID-19 64 patient samples using various conventional RNA-seq sequencing library construction 65 methods. Although these library construction methods successfully generated a draft 66 genome, several drawbacks hinder the use of these methods for routine viral genome 67 sequencing from the surge of clinical samples during an outbreak. 68 69 One direct library construction approach which was used to generate the SARS-CoV-2 70 draft genome(1, 2) essentially captures each sample's entire metatranscriptome, in which 71 SARS-CoV-2 is just one species ...

One viral sequence for each host? – The neglected within-host diversity as the main stage of SARS-CoV-2 evolution

“…Viral RNA was extracted using the QIAamp® ViralRNA Mini Kit according to the manufacturer's instructions. After performing rRNA removal using the MGIEasy rRNA Depletion Kit (BGI, Shenzhen, China), we used the novel Metagenomic RNA (CHEN et al 2020). The final viral-enriched libraries were sequenced on an Illumina NextSeq500 in 2×75bp pair-end mode.…”

Dynamics of the Upper Respiratory Tract Microbiota and Its Association with Fatality in COVID-19 Patients

“…The RNA was converted into Illumina-compatible sequencing libraries with MINERVA protocol 46 . Brie y, ve μl of nucleic acid was digested with DNase I (New England Biolabs, US), and then the rst-strand cDNA was synthesized with Oligo dT primer (5'-T30VN-3'), random decamer primer (5'-N10-3'), and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA).…”

COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

“…Various NGS based approaches have been developed to perform SARS-CoV-2 WGS using different sequencing platforms. These include direct RNA sequencing and metagenomics [12][13][14] , amplicon-based methods 13,15,16 , and oligonucleotide capture-based methods 13,[16][17][18] . Recently, numerous commercial kits based on the above methods have become available on the market and are being deployed in SARS-CoV-2 genomic surveillance 19 .…”