HiDRA-seq: High-Throughput SARS-CoV-2 Detection by RNA Barcoding and Amplicon Sequencing

Abstract

10The recent outbreak of a new coronavirus that causes a Severe Acute Respiratory Syndrome in humans 11 (SARS-CoV-2) has developed into a global pandemic with over 6 million reported cases and more than 12 375,000 deaths worldwide. Many countries have faced a shortage of diagnostic kits as well as a lack 13 of infrastructure to perform necessary testing. Due to these limiting factors, only patients showing 14 symptoms indicating infection were subjected to testing, whilst asymptomatic individuals, who are 15 widely believed to be responsible for the fast dispersion of the virus, were largely omitted from the 16 testing regimes. The inability to implement high throughput diagnostic and contact tracing strategies 17 has forced many countries to institute lockdowns with severe economic and social consequences. The 18World Health Organization (WHO) has encouraged affected countries to increase testing capabilities 19 to identify new cases, allow for a well-controlled lifting of lockdown measures, and prepare for future 20 outbreaks. Here, we propose HiDRA-seq, a rapidly implementable, high throughput, and scalable 21 solution that uses NGS lab infrastructure and reagents for population-scale SARS-CoV-2 testing. This 22 method is based on the use of indexed oligo-dT primers to generate barcoded cDNA from a large 23 number of patient samples. From this, highly multiplexed NGS libraries are prepared targeting SARS-24CoV-2 specific regions and sequenced. The low amount of sequencing data required for diagnosis 25 allows the combination of thousands of samples in a sequencing run, while reducing the cost to 26 approximately 2 CHF/EUR/USD per RNA sample. Here, we describe in detail the first version of the 27 protocol, which can be further improved in the future to increase its sensitivity and to identify other 28 respiratory viruses or analyze individual genetic features associated with disease progression. 29 30

Initial insights into the genetic epidemiology of SARS-CoV-2 isolates from Kerala suggest local spread from limited introductions

“…The availability of high-throughput sequencing approaches has enabled researchers to sequence genomes as the pandemic progressed in their respective countries. A number of methods have been adopted for rapid high throughput sequencing of SARS-CoV-2 including shotgun sequencing [4] , PCR amplicon, and hybridization/capture-based enrichment and sequencing [5][6][7] .…”

“…A number of approaches have emerged for rapid and scalable sequencing of SARS-CoV-2 from clinical isolates. This includes direct shotgun approaches as well as targeted amplicon-based and targeted capture-based approaches [4][5][6] . Sequencing based approaches provide a unique opportunity for high fidelity of detection and for understanding the genetic epidemiology of SARS-CoV-2 [7] .…”

INSIGHT: a population scale COVID-19 testing strategy combining point-of-care diagnosis with centralised high-throughput sequencing

“…While different research groups [14,15] and companies (e.g. COVIDSeq [26] from Illumina and SwabSeq [27] from Octant) have proposed to use NGS to expand testing capacity, most of them are RT-PCR-based, restricting amplification and barcoding to centralised facilities.…”

“…However, it is difficult to control for patient operational error and it is also challenging for centralised data collection. Centralised testing using a next generation sequencing (NGS) readout [14,15] has also been proposed, which allows efficient scaling of testing and simple data collection, but patients do not have immediate access to the testing results thus delaying the early isolation of pre-symptomatic or asymptomatic patients. Here we propose INSIGHT (Isothermal NASBA-Sequencing-based hIGH-througput Test): a two-stage testing strategy, using a combination of isothermal Nucleic Acid Sequence-Based Amplification (NASBA) and NGS technologies, combining the advantages of near-patient and centralised testing ( Figure 1a).…”

McQ – An open-source multiplexed SARS-CoV-2 quantification platform

“…Reads mapping to viral sequences in samples containing no SARS-CoV-2 RNA are a source of false positive test outcomes and limit sensitivity of detection assays. Non-zero background has been observed in other sequencing-based assays for SARS-CoV-2 detection 23,24,53 , and molecular contamination and mis-assignment of sequencing reads were identified as main underlying sources 23 . Leveraging the UMI information we have shown that between 1% and 6% of the UMI counts in a pool are contaminants of another pool (Figure S7B), resulting from events taking place after reverse transcription (during library amplification or sequencing).…”