A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva
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Evaluation Summary:
The authors developed an approach for surveillance screening for SARS-CoV-2, which involves the isothermic amplification of a region of the SARS-CoV-2 nucleocapsid gene using RT-LAMP, followed by detection with deep sequencing. High-throughput and cost effectiveness is achieved by two sets of barcodes that allow up to about 37,000 samples to be combined into one deep sequencing run. Moreover, the authors demonstrate they can do the detection from saliva collected on paper, which should make sample collection easier. The main strength of the work lies in solving the technical aspects for the approach to work. The main weakness is that real-world high-throughput detection is not conclusively demonstrated as only 8 clinical saliva samples are examined.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The COVID-19 pandemic has created an urgent need for rapid, effective, and low-cost SARS-CoV-2 diagnostic testing. Here, we describe COV-ID, an approach that combines RT-LAMP with deep sequencing to detect SARS-CoV-2 in unprocessed human saliva with a low limit of detection (5–10 virions). Based on a multi-dimensional barcoding strategy, COV-ID can be used to test thousands of samples overnight in a single sequencing run with limited labor and laboratory equipment. The sequencing-based readout allows COV-ID to detect multiple amplicons simultaneously, including key controls such as host transcripts and artificial spike-ins, as well as multiple pathogens. Here, we demonstrate this flexibility by simultaneous detection of 4 amplicons in contrived saliva samples: SARS-CoV-2, influenza A, human STATHERIN , and an artificial SARS calibration standard. The approach was validated on clinical saliva samples, where it showed excellent agreement with RT-qPCR. COV-ID can also be performed directly on saliva absorbed on filter paper, simplifying collection logistics and sample handling.
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Author Response:
Reviewer #1:
In this work Warneford-Thomson et al. developed an approach for surveillance screening for SARS-CoV-2, which involves the isothermic amplification of a region of the SARS-CoV-2 nucleocapsid gene using RT-LAMP, followed by detection with deep sequencing. High-throughput and cost effectiveness is achieved by two sets of barcodes that allow up to about 37,000 samples to be combined into one deep sequencing run. Moreover, the authors demonstrate they can do the detection from saliva collected on paper, which should make sample collection easier.
The main strength of the work lies in the technical aspects, including setting up multiple controls such as a detection of a human gene, and multiplexing with detection of the influenza virus.
The main weakness is that there are multiple other papers either …
Author Response:
Reviewer #1:
In this work Warneford-Thomson et al. developed an approach for surveillance screening for SARS-CoV-2, which involves the isothermic amplification of a region of the SARS-CoV-2 nucleocapsid gene using RT-LAMP, followed by detection with deep sequencing. High-throughput and cost effectiveness is achieved by two sets of barcodes that allow up to about 37,000 samples to be combined into one deep sequencing run. Moreover, the authors demonstrate they can do the detection from saliva collected on paper, which should make sample collection easier.
The main strength of the work lies in the technical aspects, including setting up multiple controls such as a detection of a human gene, and multiplexing with detection of the influenza virus.
The main weakness is that there are multiple other papers either published or archived that use RT-LAMP for SARS-CoV-2 detection, deep sequencing for SARS-CoV-2 detection, or both. These are cited in the current work, which is very well written and presented. Whether this method is better than the others which have the same aim of developing cost-effective and high-throughput detection is not conclusively demonstrated as only 8 clinical saliva samples are examined.
We do not wish to claim that our method is better than the others. We think it has advantages and disadvantages and certainly it should be further optimized before scaling it up to population level. We have added these considerations to the text (lines 376–80).
Furthermore, the requirement for deep sequencing and batching many samples for cost-effectiveness will, in most situations, greatly increase turn-around time. This will make surveillance much less effective, since by the time results are fed back, the asymptomatically infected individual would have had more opportunity to transmit the infection to others.
We argue that time from sample to result is a mostly a function of logistics and not of the method. With proper set ups the time from sample collection to results could be < 16 hours, which would be compatible with population-level surveillance. We added these considerations to the text.
However, the deep sequencing step may be very useful for surveillance of circulating SARS-CoV-2 spike sequences to detect emerging variants within a population, provided this method can be modified to do it.
We agree and we mention this possibility in the discussion.
Reviewer #2:
In 'COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva', Warneford-Thomson et al. present a novel methodology to perform large numbers of COVID-19 tests in parallel. Their approach takes unprocessed saliva and requires only a small number of experimental steps before the results are sequenced overnight to generate many thousands of results. This straightforward experimental design should allow the protocol to be expanded to a number of settings where population-level monitoring is required in order to contain outbreaks and reduce transmission. In this paper, the authors demonstrate the efficacy of their approach and perform a large number of benchmarking experiments to quantify its sensitivity, specificity and limitations of detection. They are able to detect artificially created infections (spike-ins) with as low as 5 virions per µL and all clinically available samples agreed with the standard RT-qPCR test. This method can detect both SARS-CoV-2 and Influenza infection and can also be applied to saliva samples which have been collected on filter paper, a strategy which will further simplify the testing regime.
The authors have spent much time testing this approach but these have largely been limited to analysing artificially created infections. The only results which were obtained were from eight clinically derived samples which are presented in Figure 2E. Although all results from this approach agreed with the standard clinical test this is a small number of tests compared to the total number of tests which are reported in this paper. It is also only a small proof-of-principle experiment to justify a quick rollout of this technology.
We have now performed COV-ID on 120 additional patient samples (new Figure 2-figure supplement 2). These new results are described in the text.
The potential for this technology to perform rapid, high-throughput SARS-CoV-2 testing alongside the potential for very low sequencing costs (Figure 4G) is impressive. It is noted in the manuscript that this will require 96 unique barcodes but only 32 are tested here. All but three of these 32 work for the SARS-CoV-2 N2 primers and required STATH control but how will the remaining 67 primers be derived (i.e. is it realistic that this can be made to work to deliver the promise of this approach)?
The current COV-ID patient barcodes are 5 base pairs long. This allows for 4^5 = 1,024 combinations. Out of an abundance of caution, we excluded barcodes with homology to the reverse complement of the RT-LAMP primers used in any of the experiments (i.e. primers for SARS-CoV-2 N2, STATHERIN, ACTIN, and influenza virus) and then selected a set of 32 with Hamming distances of at least 2 from each other. This is now described more in detail in the methods.
Regarding the numbers, out of 1,024 5-bp barcodes, 404 were removed due to homology, leaving 620. Of these, we could find at least 163 with Hamming distance ≥ 2 from each other. Even with a substantial failure rate, this should allow for 96 working barcodes. If we had only considered clashes with N2 and STATHERIN primers, the number of available barcodes would be substantially higher.
Overall, this is an interesting paper which has very clear real-world application to helping to defeat the ongoing COVID-19 pandemic, but some extra validations are needed to fully demonstrate its performance in clinical and/or public health settings.
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Evaluation Summary:
The authors developed an approach for surveillance screening for SARS-CoV-2, which involves the isothermic amplification of a region of the SARS-CoV-2 nucleocapsid gene using RT-LAMP, followed by detection with deep sequencing. High-throughput and cost effectiveness is achieved by two sets of barcodes that allow up to about 37,000 samples to be combined into one deep sequencing run. Moreover, the authors demonstrate they can do the detection from saliva collected on paper, which should make sample collection easier. The main strength of the work lies in solving the technical aspects for the approach to work. The main weakness is that real-world high-throughput detection is not conclusively demonstrated as only 8 clinical saliva samples are examined.
(This preprint has been reviewed by eLife. We include the public …
Evaluation Summary:
The authors developed an approach for surveillance screening for SARS-CoV-2, which involves the isothermic amplification of a region of the SARS-CoV-2 nucleocapsid gene using RT-LAMP, followed by detection with deep sequencing. High-throughput and cost effectiveness is achieved by two sets of barcodes that allow up to about 37,000 samples to be combined into one deep sequencing run. Moreover, the authors demonstrate they can do the detection from saliva collected on paper, which should make sample collection easier. The main strength of the work lies in solving the technical aspects for the approach to work. The main weakness is that real-world high-throughput detection is not conclusively demonstrated as only 8 clinical saliva samples are examined.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this work Warneford-Thomson et al. developed an approach for surveillance screening for SARS-CoV-2, which involves the isothermic amplification of a region of the SARS-CoV-2 nucleocapsid gene using RT-LAMP, followed by detection with deep sequencing. High-throughput and cost effectiveness is achieved by two sets of barcodes that allow up to about 37,000 samples to be combined into one deep sequencing run. Moreover, the authors demonstrate they can do the detection from saliva collected on paper, which should make sample collection easier.
The main strength of the work lies in the technical aspects, including setting up multiple controls such as a detection of a human gene, and multiplexing with detection of the influenza virus.
The main weakness is that there are multiple other papers either published or …
Reviewer #1 (Public Review):
In this work Warneford-Thomson et al. developed an approach for surveillance screening for SARS-CoV-2, which involves the isothermic amplification of a region of the SARS-CoV-2 nucleocapsid gene using RT-LAMP, followed by detection with deep sequencing. High-throughput and cost effectiveness is achieved by two sets of barcodes that allow up to about 37,000 samples to be combined into one deep sequencing run. Moreover, the authors demonstrate they can do the detection from saliva collected on paper, which should make sample collection easier.
The main strength of the work lies in the technical aspects, including setting up multiple controls such as a detection of a human gene, and multiplexing with detection of the influenza virus.
The main weakness is that there are multiple other papers either published or archived that use RT-LAMP for SARS-CoV-2 detection, deep sequencing for SARS-CoV-2 detection, or both. These are cited in the current work, which is very well written and presented. Whether this method is better than the others which have the same aim of developing cost-effective and high-throughput detection is not conclusively demonstrated as only 8 clinical saliva samples are examined.
Furthermore, the requirement for deep sequencing and batching many samples for cost-effectiveness will, in most situations, greatly increase turn-around time. This will make surveillance much less effective, since by the time results are fed back, the asymptomatically infected individual would have had more opportunity to transmit the infection to others. However, the deep sequencing step may be very useful for surveillance of circulating SARS-CoV-2 spike sequences to detect emerging variants within a population, provided this method can be modified to do it.
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Reviewer #2 (Public Review):
In 'COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva', Warneford-Thomson et al. present a novel methodology to perform large numbers of COVID-19 tests in parallel. Their approach takes unprocessed saliva and requires only a small number of experimental steps before the results are sequenced overnight to generate many thousands of results. This straightforward experimental design should allow the protocol to be expanded to a number of settings where population-level monitoring is required in order to contain outbreaks and reduce transmission. In this paper, the authors demonstrate the efficacy of their approach and perform a large number of benchmarking experiments to quantify its sensitivity, specificity and limitations of detection. They …
Reviewer #2 (Public Review):
In 'COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva', Warneford-Thomson et al. present a novel methodology to perform large numbers of COVID-19 tests in parallel. Their approach takes unprocessed saliva and requires only a small number of experimental steps before the results are sequenced overnight to generate many thousands of results. This straightforward experimental design should allow the protocol to be expanded to a number of settings where population-level monitoring is required in order to contain outbreaks and reduce transmission. In this paper, the authors demonstrate the efficacy of their approach and perform a large number of benchmarking experiments to quantify its sensitivity, specificity and limitations of detection. They are able to detect artificially created infections (spike-ins) with as low as 5 virions per µL and all clinically available samples agreed with the standard RT-qPCR test. This method can detect both SARS-CoV-2 and Influenza infection and can also be applied to saliva samples which have been collected on filter paper, a strategy which will further simplify the testing regime.
The authors have spent much time testing this approach but these have largely been limited to analysing artificially created infections. The only results which were obtained were from eight clinically derived samples which are presented in Figure 2E. Although all results from this approach agreed with the standard clinical test this is a small number of tests compared to the total number of tests which are reported in this paper. It is also only a small proof-of-principle experiment to justify a quick rollout of this technology.
The potential for this technology to perform rapid, high-throughput SARS-CoV-2 testing alongside the potential for very low sequencing costs (Figure 4G) is impressive. It is noted in the manuscript that this will require 96 unique barcodes but only 32 are tested here. All but three of these 32 work for the SARS-CoV-2 N2 primers and required STATH control but how will the remaining 67 primers be derived (i.e. is it realistic that this can be made to work to deliver the promise of this approach)?
Overall, this is an interesting paper which has very clear real-world application to helping to defeat the ongoing COVID-19 pandemic, but some extra validations are needed to fully demonstrate its performance in clinical and/or public health settings.
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SciScore for 10.1101/2021.04.23.21255523: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Human samples: Clinical saliva samples used for Fig. 2E were obtained and characterized as part of a separate study at the University of Pennsylvania44 and collected under Institutional Review Board (IRB)-approved protocols (IRB protocol #842613 and #813913).
Consent: After verbal consent was obtained by a trained research coordinator, patients were instructed to self-collect saliva into a sterile specimen container which was then placed on ice until further processing for analysis.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources After testing, we determined that … SciScore for 10.1101/2021.04.23.21255523: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Human samples: Clinical saliva samples used for Fig. 2E were obtained and characterized as part of a separate study at the University of Pennsylvania44 and collected under Institutional Review Board (IRB)-approved protocols (IRB protocol #842613 and #813913).
Consent: After verbal consent was obtained by a trained research coordinator, patients were instructed to self-collect saliva into a sterile specimen container which was then placed on ice until further processing for analysis.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources After testing, we determined that 12 nts and 11 nts were most effective for the P5 and P7 binding regions, respectively, being the shortest insertion that allowed reliable PCR amplification from LAMP products without impacting LAMP efficiency. LAMPsuggested: (LAMP, RRID:SCR_001740)Sequencing: Libraries were sequenced on one of the following Illumina instruments: MiSeq, NextSeq 500, NextSeq 550, NovaSeq 6000 and sequenced using single end programs with a minimum of 40 cycles on Read 1 and 8 cycles for index 1 (on P7) and index 2 (on P5). MiSeqsuggested: (A5-miseq, RRID:SCR_012148)Sequence Analysis: Reads were filtered for optical quality using FASTX-toolkit utility fastq_quality_filter (http://hannonlab.cshl.edu/fastx_toolkit/), then cutadapt45 was used to remove adapters and demultiplex LAMP barcodes. http://hannonlab.cshl.edu/fastx_toolkit/suggested: (FASTX-Toolkit, RRID:SCR_005534)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Because of reagent limitations and diagnostic testing bottlenecks, prioritization of COVID diagnostic testing continues to be for symptomatic individuals and individuals who are particularly vulnerable for infection after exposure41. Private organizations, including colleges and universities, have circumvented some of these challenges by contracting with private laboratories to establish asymptomatic surveillance testing protocols; this is a costly option for population-level surveilling of asymptomatic SARS-CoV-2 infections. Several effective COVID-19 vaccines have been developed and there is a concerted ongoing global vaccination effort, providing a concrete means to end the pandemic. Despite this progress there are several potential risks that require vigilance: possible COVID-19 transmission in vaccinated individuals, emergence of vaccine-resistant viral variants, and public skepticism of vaccines or faltering compliance with social distancing guidelines42. For these reasons ongoing testing and surveillance efforts will remain important for the foreseeable future, both to monitor the progress of vaccination in reducing symptomatic cases and to detect emerging variants. In order to scale testing to an effective volume and frequency, surveillance tests must demonstrate the following qualities: 1) sensitivity, to identify both asymptomatic and symptomatic carriers; 2) simplicity in methodology, to be performed in a number of traditional diagnostic laboratories, without speci...
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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