Early sample tagging and pooling enables simultaneous SARS-CoV-2 detection and variant sequencing

Abstract

Most SARS-CoV-2 diagnostic tests have relied on RNA extraction followed by quantitative reversetranscription PCR assays (RT-qPCR). Whereas automation improved logistics and different pooling strategies increased testing capacity, highly multiplexed next generation sequencing (NGS) diagnostics remain a largely untapped resource. NGS tests have the potential to dramatically increase throughput while providing crucial SARS-CoV-2 variant information. Current NGS-based detection and genotyping assays for SARS-CoV-2 are costly, mostly due to parallel sample processing through multiple steps. Here, we have established ApharSeq, in which samples are barcoded in the lysis buffer and pooled prior to reverse transcription. We validated this assay by applying ApharSeq to more than 500 clinical samples from the Clinical Virology Lab at Hadassah hospital in a robotic workflow. The assay was linear across five orders of magnitude, and the limit of detection was Ct 33 (~1000 copies/ml, 95% sensitivity) with >99.5% specificity. ApharSeq provided targeted high-confidence genotype information due to unique molecular identifiers incorporated into this method. Due to early pooling, we were able to estimate a 10-fold to a 100fold reduction in labor, automated liquid handling, and reagent requirements in high throughput settings compared to current testing methods. The protocol can be tailored to assay other host or pathogen RNA targets simultaneously. These results suggest that ApharSeq can be a promising tool for current and future mass diagnostic challenges.

A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

“…Several methods have emerged that harness massively parallel next generation sequencing for diagnostics of SARS-CoV-2 ( Simonetti et al, 2021 ; Bloom et al, 2021 ; Yelagandula et al, 2021 ; Aynaud et al, 2021 ; Wu et al, 2021 ; de Mello Malta et al, 2021 ; Chappleboim et al, 2021 ; Peto et al, 2021 ; Dao Thi et al, 2020 ; Ludwig et al, 2021 , Credle et al, 2021 ), reflecting the desire for novel approaches to address the shortcomings of labor-intensive individual clinical diagnostic testing. COV-ID complements these approaches by providing a method that can screen thousands of individuals with a heated incubator, a single PCR thermocycler and access to a sequencer.…”

“…While effective, these methods rely on individual PCR amplification of each patient sample, thus requiring a large number of thermal cyclers for massive scale-up. An alternative approach, ApharSeq, addresses this bottleneck by annealing barcoded RT primers to viral RNA and pooling samples prior to amplification, but the need for specialized oligo-dT magnetic beads might constitute a separate adoption barrier for this method ( Chappleboim et al, 2021 ). Finally, methods have been designed to take advantage of the extreme sensitivity and isothermal conditions of loop-mediated isothermal amplification (LAMP) ( Peto et al, 2021 ; Dao Thi et al, 2020 ; Ludwig et al, 2021 ), but they require additional manipulation to introduce barcodes ( Peto et al, 2021 ; Dao Thi et al, 2020 ) or do not allow for convenient multiplexing ( Ludwig et al, 2021 ).…”

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern

“…On the basis of the genomic sequences, different techniques have been developed for the detection of variants. The main features of three groups of techniques, including sequencing [ [93] , [94] , [95] , [96] , [97] ], mutation-specific RT-PCR [ [98] , [99] , [100] ], and CRISPR-based methods, are summarized and compared ( Table S1 ). In general, sequencing-based methods can provide high-resolution information of the whole genome or a particular gene, enabling the identification of new variants.…”

Evaluation of immunoprotection against coronavirus disease 2019: Novel variants, vaccine inoculation, and complications

“…High-throughput sequencing (HTS) produces full-length genome sequences [ 10 ], allowing identification of viral variants. Although, HTS is a powerful technique for identifying the increasingly variating SARS-CoV-2 genome [ 11 ], its high cost prevents its popularization.

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