ApharSeq: An Extraction-free Early-Pooling Protocol for Massively Multiplexed SARS-CoV-2 Detection

Abstract

The global SARS-CoV-2 pandemic led to a steep increase in the need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays that involve a separate RNA extraction and qPCR reaction for each sample with a fixed cost and reaction time. While automation and improved logistics can increase the capacity of these tests, they cannot exceed this lower bound dictated by one extraction and reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information on viral strains and host immune response. Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq, includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. We characterize the quantitative aspects of the assay, and test ApharSeq on dozens of clinical samples in a robotic workflow. Our proposed workflow is estimated to reduce costs by 10-50 fold, labor by 5-100 fold, automated liquid handling by 5-10 fold, and reagent requirements by 100-1000 fold compared to existing testing methods.

LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding

“…The current gold standard diagnostic test for detection of active SARS-CoV-2 infection is viral RNA extraction from a biospecimen followed by RT-qPCR to amplify and detect conserved regions of the SARS-CoV-2 genome. With increasing infection numbers, this has been complemented by antigen-based tests, which provide rapid results but have limited sensitivity 6 , and sequencing-based approaches, which have increased throughput but still require RNA extraction and/or thermocycling devices [7][8][9][10][11] . Here we describe LAMP-Seq, an approach that combines RT-LAMP 12,13 with molecular barcoding to detect viral genomes in unpurified lysates at high throughput.…”

Unified platform for genetic and serological detection of COVID-19 with single-molecule technology

“…Recently, many new diagnostic approaches emerged to tackle the increasing need for better and diverse methods [2]. Those include reverse transcription coupled with nanopore sensing [3], isothermal amplification [4][5][6], CRISPR based methods [7,8], next generation sequencing based methods [9] and improvement of RT-PCR timing through plasmonic thermocycling [10]. These novel approaches improve the time, costs and accessibility of the tests, although still mostly rely on enzymatic processes.…”

Unified platform for genetic and serological detection of COVID-19 with single-molecule technology

“…Recently, many new diagnostic approaches emerged to tackle the increasing need for better and diverse methods [2]. Those include reverse transcription coupled with nanopore sensing [3], isothermal amplification [4-6], CRISPR based methods [7, 8], next generation sequencing based methods [9] and improvement of RT-PCR timing through plasmonic thermocycling [10, 11]. These novel approaches improve the time, costs and accessibility of the tests, although still mostly rely on enzymatic processes.…”