A rapid, low-cost, and highly sensitive SARS-CoV-2 diagnostic based on whole-genome sequencing
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Abstract
Early detection of SARS-CoV-2 infection is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset. Here, we introduce a low-cost, high-throughput method for diagnosing and studying SARS-CoV-2 infection. Dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), this method amplifies the entirety of the SARS-CoV-2 genome. This contrasts with typical RT-PCR-based diagnostic tests, which amplify only a few loci. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network ( https://artic.network/ ) with short-read DNA sequencing and de novo genome assembly. Using this method, we can reliably (>95% accuracy) detect SARS-CoV-2 at a concentration of 84 genome equivalents per milliliter (GE/mL). The vast majority of diagnostic methods meeting our analytical criteria that are currently authorized for use by the United States Food and Drug Administration with the Coronavirus Disease 2019 (COVID-19) Emergency Use Authorization require higher concentrations of the virus to achieve this degree of sensitivity and specificity. In addition, we can reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy given sufficient viral load. The genotypic data in these genome assemblies enable the more effective analysis of disease spread than is possible with an ordinary binary diagnostic. These data can also help identify vaccine and drug targets. Finally, we show that the diagnoses obtained using POLAR of positive and negative clinical nasal mid-turbinate swab samples 100% match those obtained in a clinical diagnostic lab using the Center for Disease Control’s 2019-Novel Coronavirus test. Using POLAR, a single person can manually process 192 samples over an 8-hour experiment at the cost of ~$36 per patient (as of December 7 th , 2022), enabling a 24-hour turnaround with sequencing and data analysis time. We anticipate that further testing and refinement will allow greater sensitivity using this approach.
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SciScore for 10.1101/2020.04.25.061499: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Collection of SARS-CoV-2: The quantified sample material used for the limit of detection was genomic RNA (gRNA) extracted from a cell line (Vero E6, ATCC® CRL-1586™) infected with SARS-related coronavirus 2 (SARS-CoV-2, isolate USA-WA1/2020, Lot: 70033700), using QIAamp® Viral RNA Mini Kit (Qiagen 52904) deposited by American Type Culture Collection (ATCC) and obtained from Biodefense and Emerging Infections Research Resources … SciScore for 10.1101/2020.04.25.061499: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Collection of SARS-CoV-2: The quantified sample material used for the limit of detection was genomic RNA (gRNA) extracted from a cell line (Vero E6, ATCC® CRL-1586™) infected with SARS-related coronavirus 2 (SARS-CoV-2, isolate USA-WA1/2020, Lot: 70033700), using QIAamp® Viral RNA Mini Kit (Qiagen 52904) deposited by American Type Culture Collection (ATCC) and obtained from Biodefense and Emerging Infections Research Resources Repository (BEI Resources). Vero E6suggested: NoneNegative control RNA extraction: Approximately 1 million K562 cells and 1 million HeLa cells cultured in our lab were used as the starting material for RNA extraction using columns provided in the RNeasy Mini Kit (Cat no: 74104). K562suggested: NoneHeLasuggested: NoneSoftware and Algorithms Sentences Resources SARS-CoV-2 Coverage Analysis: To compare SARS-CoV-2 coverage across starting concentrations, FASTQs were aligned to the SARS-CoV-2 reference genome (NCBI Reference Sequence: NC_045512.2) using BWA66 with default parameters. BWA66suggested: NoneComparative Assembly Statistics: In order to determine the accuracy of our de novo assemblies, we compared our SARS-CoV-2 de novo assembly to the SARS-CoV-2 reference assembly (NCBI Reference Sequence: NC_045512.2), our Human coronavirus 229E de novo assembly to the Human coronavirus 229E reference assembly (NCBI Reference Sequence: NC_002645.1), our Avian Coronavirus to the Avian Coronavirus Massachusetts (formerly Avian Infectious Bronchitis Virus) (GenBank: GQ504724.1), our Human Coronavirus NL63 de novo assembly to the Human Coronavirus NL63 (GenBank: AY567487.2) reference assembly and our Porcine Respiratory Virus to the PRCV ISU-1 (GenBank: DQ811787.1) reference genome using MetaQuast73. MetaQuast73suggested: NoneResults from OddPub: Thank you for sharing your code.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:At the same time, the approach we describe also has several limitations as compared to other diagnostic tests. For example, our method does not provide any information regarding the SARS-CoV-2 viral load of a patient. This might be addressed by adding a synthetic RNA molecule with a known concentration into each patient sample in order to estimate viral load using relative coverage. Another limitation is that our method is slower than other approaches, in the sense that it requires 24 hours from acquisition of a patient sample to a diagnostic result. By contrast, Abbott Labs (one example of many new diagnostic technologies developed in the past few months) has developed a diagnostic test capable of returning results in as little as 5 minutes for a positive result and 13 minutes for a negative result57. However, it is worth noting that the maximum number of diagnostic results an Abbot device could complete running 24 hours a day is roughly between 111 tests and 126 tests depending on the number of positive results58,59. Beyond diagnosis of individual patients, POLAR can also be applied to SARS-CoV-2 surveillance, in settings such as municipal wastewater treatment plants60. In principle, such approaches could identify and characterize infection in a neighborhood or city very inexpensively, even for a large population, informing public policy decisions. We note that multiple groups have been developing methods for sequencing whole SARS-CoV-2 genomes, and in some cases sharing th...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.04.25.061499: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding We used POLAR to classify 5 positive and 5 negative clinical samples in a blinded experiment , exhibiting 100 % agreement with the CDC 2019-Novel Coronavirus test . Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources We also prepared a series of negative controls: 2 replicates of nuclease-free water , processed separately from the positive samples; 2 replicates of HeLa RNA extract , and 2 replicates of K562 RNA extract . HeLasuggested: None…SciScore for 10.1101/2020.04.25.061499: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding We used POLAR to classify 5 positive and 5 negative clinical samples in a blinded experiment , exhibiting 100 % agreement with the CDC 2019-Novel Coronavirus test . Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources We also prepared a series of negative controls: 2 replicates of nuclease-free water , processed separately from the positive samples; 2 replicates of HeLa RNA extract , and 2 replicates of K562 RNA extract . HeLasuggested: NoneMethods & Materials Collection of SARS-CoV-2 The quantified sample material used for the limit of detection was genomic RNA ( gRNA ) extracted from a cell line ( Vero E6 , ATCC® CRL-1586™ ) infected with SARS-related coronavirus 2 ( SARSCoV-2 , isolate USA-WA1/2020 , Lot: 70033700) , using QIAamp® Viral RNA Mini Kit ( Qiagen 52904 ) deposited by American Type Culture Collection ( ATCC ) and obtained from Biodefense and Emerging Infections Research Resources Repository ( BEI Resources) . Vero E6suggested: NoneNegative control RNA extraction Approximately 1 million K562 cells and 1 million HeLa cells cultured in our lab were used as the starting material for RNA extraction using columns provided in the RNeasy Mini Kit ( Cat no: 74104) . K562suggested: NoneSoftware and Algorithms Sentences Resources For instance , when examining the 21 different publicly available SARS-CoV-2 RT-PCR primer sets from the UCSC Genome Browser , we see that , even in aggregate , these primers amplify only 6.86 % of the SARSCoV-2 genome. UCSC Genome Browsersuggested: (UCSC Genome Browser, SCR_005780)For each library , we generated a de novo assembly using the memory efficient assembly algorithm MEGAHIT51 with default parameters . MEGAHIT51suggested: NoneBy contrast , Abbott Labs ( one example of many new diagnostic technologies developed in the past few months ) has developed a diagnostic test capable of returning results in as little as 5 minutes for a positive result and 13 minutes for a negative result57 . Abbott Labssuggested: NoneSARS-CoV-2 Coverage Analysis To compare SARS-CoV-2 coverage across starting concentrations, FASTQs were aligned to the SARS-CoV-2 reference genome (NCBI Reference Sequence: NC_045512.2) using BWA66 with default parameters. BWA66suggested: NoneComparative Assembly Statistics In order to determine the accuracy of our de novo assemblies, we compared our SARS-CoV-2 de novo assembly to the SARS-CoV-2 reference assembly (NCBI Reference Sequence: NC_045512.2), our Human coronavirus 229E de novo assembly to the Human coronavirus 229E reference assembly (NCBI Reference Sequence: NC_002645.1), our Avian Coronavirus to the Avian Coronavirus Massachusetts (formerly Avian Infectious Bronchitis Virus) (GenBank: GQ504724.1), our Human Coronavirus NL63 de novo assembly to the Human Coronavirus NL63 (GenBank: AY567487.2) reference assembly and our Porcine Respiratory Virus to the PRCV ISU1 (GenBank: DQ811787.1) reference genome using MetaQuast73. MetaQuast73suggested: NoneResults from OddPub: Thank you for sharing your code.
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SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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