Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow

Abstract

Since the first reported case of the new coronavirus infection in Wuhan, China, researchers and governments have witnessed an unseen rise in the number of cases. Thanks to the rapid work of Chinese scientists, the pathogen now called SARS-CoV-2 has been identified and its whole genome was deposited in public databases by early January 2020. The availability of the genome has allowed researchers to develop Reverse Transcription—Polymerase Chain Reaction (RT-PCR) assays, which are now the gold-standard for molecular diagnosis of the respiratory syndrome COVID19. Because of the rising number of cases and rapid spreading, the world has been facing a shortage of RT-PCR supplies, especially the ones involved in RNA extraction. This has been a major bottleneck to increase testing capacity in many countries that do not significantly manufacture these supplies, such as Brazil. Additionally, RT-qPCR scalability is highly dependent on equipment that usually performs testing of 96 samples at a time. In this work, we describe a cost-effective molecular NGS-based test for diagnosis of COVID19, which uses a single-step RNA extraction and presents high scalability and accuracy when compared to the gold-standard RT-qPCR. A single run of the NGS-based test using the Illumina NextSeq 550 mid-end sequencing equipment is able to multiplex 1,536 patient’s samples, providing individual semi-qualitative results (detected, not detected). Detected results are provided with fragments per million (FPM) values, which was demonstrated to correlate with RT-qPCR Cycle Threshold (CT) values. Besides, usage of the high-end Illumina Novaseq platform may yield diagnostic for up to 6144 samples in a single run. Performance results when compared with RT-qPCR show general accuracy of 96%, and 98% when only samples with CT values (gene N) lower than 30 are considered. We have also developed an online platform, termed VarsVID, to help test executors to easily scale testing numbers. Sample registering, wet-lab worksheets generation, sample sheet for sequencing and results’ display are all features provided by VarsVID. Altogether, these results will contribute to control COVID19 pandemics.

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

“…We observed consistent amplification for all seven SARS-CoV-2 lineages with 10,000 copies of target input per reaction (200,000 copies/mL) (Fig. 1E), which is comparable to the target input of >200,000 copies/mL viruses (<30 Ct value) required for sequencing workflows used in SARS-CoV-2 variant surveillance (32,33).…”

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.…”

“…In general, sequencing-based protocols use libraries of amplification primers to tag reads originating from each individual patient sample with a unique index that can be identified and deconvoluted after sequencing, thus allowing pooling of tens of thousands of samples in a single assay. Several methods, including SARSeq, SPAR-Seq, Swab-seq, COVseq, and INSIGHT directly amplify the viral RNA by RT-PCR and simultaneously introduce barcodes ( 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 ). 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.…”

Clinical Evaluation of a Multiplex PCR Assay for Simultaneous Detection of 18 Respiratory Pathogens in Patients with Acute Respiratory Infections

“…On the one hand, the diagnostic sensitivity and specificity of the MPA assay are lower than those of NGS. As a robust tool to obtain extensive genetic information, NGS is a more sensitive, informative and expensive method for the diagnosis of various infections, with a limit of detection (LOD) values as low as 10 copies/mL for respiratory pathogens, including for challenging samples with a low viral content [ 18 ]. In the MPA assay, the LOD value was 1000 copies/mL for these 18 respiratory pathogens.…”