key: cord-0812176-scyqijkn
authors: Studer, F.; Petit, J.-L.; Engelen, S.; Mendoza-Parra, M. A.
title: Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing
date: 2021-05-22
journal: nan
DOI: 10.1101/2021.05.18.21257281
sha: fc6c1b22b5beb9df27918eb0d64bd32ad95f0de3
doc_id: 812176
cord_uid: scyqijkn

Since December 2019, the emergence of a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. As consequence, a major effort worldwide has been performed for the development of viral diagnostics strategies aiming at (i) reducing the diagnostics time, (ii) decrease the costs per assay, and (iii) providing population-scale solutions. Beyond the diagnostics requirements, the description of the B.1.1.7 strain, originated in the south of England, as a highly transmissible variant has strongly accelerated the worldwide interest in tracking SARS-CoV-2 variants occurrence. Since then, other extremely infectious variants were described and unsurprisingly further others are expected to be discovered, notably due to the long period of time on which the pandemic situation is lasting. Interestingly, all currently described SARS-CoV-2 variants present systematically several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants tracking, our proposed strategy focuses on the SPIKE region, as a way to increase the number of candidate samples to screen at once; an essential aspect to accelerate SARS-CoV-2 diagnostics, but also improve variants emergence and progression surveillance. Herein we present a proof of concept study, for performing both at once, population-scale SARS-CoV-2 diagnostics and variants tracking. This strategy relies on (i) the use of the portable and affordable MinION DNA sequencer; (ii) a DNA barcoding strategy and a SPIKE gene-centered variants tracking, for largely increasing the number of candidates per assay; and (iii) a real-time diagnostics and variants tracking monitoring thanks to our software RETIVAD. As a whole, this strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world.

Since December 2019, the emergence of a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation, leading to date to nearly 3 million deaths worldwide. Early on 2020 major viral diagnostic efforts were deployed, including the development of news strategies aiming at (i) reducing the diagnostics time, (ii) decrease the costs per assay, (iii) providing population-scale solutions, but also (iv) presenting a high sensitivity and specificity, notably to discriminate asymptomatic cases.

While costs and required time per assay were early on addressed by the development of immune-based tests 1 , their sensitivity relative to viral nucleic acid-targeting diagnostics were . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint initially questioned, but finally accepted in several countries notably due to the low throughput of diagnostics based on RT-qPCR assays 2 .

With the aim of taking advantage of the viral nucleic acid targeting diagnostics but at the same time increasing the number of candidates' through-put, strategies based on the use of massive parallel DNA sequencing for population-scale diagnostics were proposed [3] [4] [5] . These efforts rely on (i) the incorporation of DNA molecular barcodes during sample preparation; (ii) pooling barcoded samples for an "in-one-run" diagnostics assay by the use of massive parallel DNA sequencing; (iii) stratification of diagnostics' outcome with the help of bioinformatics processing. While such strategies demonstrated to up-scale the diagnostics power beyond several thousand candidates per assay, they require to count with a DNA sequencing platform where large instruments (Illumina Sequencers) are driven by specialized technical engineers. As consequence, this strategy remains applicable to highly developed countries (due to the prohibitive costs of the required DNA sequencers), which in addition requires a dedicated pipeline strategy for transporting collected samples towards sequencing centers.

Beyond the diagnostics requirements, the identification of the highly transmissible SARS-CoV-2 variant B.1.1.7 -originated in the south of England -has strongly accelerated the worldwide interest in tracking SARS-CoV-2 variants' occurrence. More recently, two other extremely infectious variants, namely the P.1 issued on the Brazilian city of Manaus and the B.1.351 initially detected In South Africa, were described, and unsurprisingly further others are expected to be discovered, notably due to the long period of time on which the pandemic situation is lasting 6 .

Herein, we describe a proof of concept study for population-scale diagnostics and variants' tracking using massive parallel DNA sequencing performed with the MinION Oxford Nanopore Technology (ONT). ONT MinION sequencers are well-known for their portability, their reduced cost, and their simplicity of use; thus, representing a suitable strategy for its implementation even in remote places over the world. In addition to a dedicated molecular biology protocol, we deliver a computational solution (RETIVAD: Real-Time Variants . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint Detector) providing the outcome of the diagnostic in a real-time mode during sequencing; allowing to earn time for diagnostics report, essential in the current context of the pandemic situation. Finally, we have also demonstrated that our strategy is able to perform SARS-CoV-2 variants' detection across multiple candidates, notably by targeting the SPIKE gene instead of covering the whole viral genome (Figure 1) . This targeted variants' tracking strategy allows increasing ~10x the potential sequencing coverage delivered during the assay, thus allowing to redistribute this resource towards the variants' screening of a large number of candidates at once.

In summary, the proposed strategy satisfies the current need for counting with a population diagnostic assay, and discriminates between the various SARS-CoV-2 variants, which are recognized to potentially present variable infection properties as well as clinical outcomes 6, 7 .

With the aim of performing SARS-CoV-2 diagnostics over multiple candidates with the MinION sequencer, we combined a regular reverse-transcription assay, with a particular PCR amplification allowing the generation of long DNA concatemers. This strategy allows to target short amplicons, like those used on quantitative real-time PCR strategies, but at the same time take advantage of the long fragments sequencing capabilities of the Oxford Nanopore technology.

Reverse transcription (RT) is performed with a Covid-19 specific primer, presenting, in addition, a unique molecular barcode (20nt length) and a common sequence adapter (Gibson sequence; 30nt length) (Figure 2A) . Hence, multiple candidate' samples are reverse-transcribed with a unique molecular barcode per candidate, allowing their identification after massive parallel DNA sequencing.

After RT assay, candidates' samples are pooled for PCR amplification into a single reaction.

For it, a second Covid-19 specific primer, presenting, in addition, a unique molecular barcode . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint (20nt length) and a reverse-oriented sequence for the common Gibson sequence adapter (30nt length) is used together with a Gibson primer (Figure 2A) . This strategy allows to incorporate a second unique molecular barcode, thus providing a combinatorial barcoding strategy per candidate which is extremely useful for increasing the number of candidates to interrogate without having to linearly increase the number of primers to purchase; an aspect which is of major relevance due to their long length (>70nt).

The presence of Gibson sequences on both extremities of the PCR amplified products allows generating concatemers during amplification (Figure 2A) . While this process takes place in an uncontrolled manner during PCR amplification, low primer concentrations were shown to give rise preferentially to long concatemers ( Figure 2B & 2C) . As consequence, concatenated PCR products can be purified from long primers (>70nt length) with SPRIselect reagent (Beckman; B23318), without the risk of losing the short amplicons.

Furthermore, the use of long concatemers increased the average sequencing coverage of MinION by a factor of ~9 (i.e. 1 million concatemers sequenced corresponds to 9 million monomers; Figure S1 and Figure 3E ), such that a large number of candidates can be potentially screened like in previous population-scale diagnostics efforts based on the use of Illumina instruments 3-5 . Overall, the proposed pipeline is composed of a standard molecular biology strategy, requiring less than 8 hours of samples preparation prior Nanopore sequencing ( Figure 2D and Methods section).

One of the major bottlenecks for population-scale Covid-19 diagnostics based on massive parallel DNA sequencing is the required time to deliver the outcome of the diagnostic. In addition to the molecular biology workflow required for samples' processing till sequencing library preparation, DNA sequencing, performed on Illumina instruments, requires an incompressible processing time, which delays downstream analyses since they can only be performed at the end of the whole sequencing process. In contrary, Oxford Nanopore . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. To validate the performance of the proposed diagnostics strategy, synthetic SARS-CoV-2 RNA control samples (Twist Biosciences) were used for mimicking variable viral RNA copies per assay. Specifically, subsequent dilutions of the synthetic Wuhan-Hu-1 (GeneBankID: MN908947.3) SARS-CoV-2 RNA control sample has been used for reverse transcription, followed by a quantitative PCR assay targeting a region on the E gene of SARS-Cov-2, defined by primer sequences described previously (E-Sarbeco) 8 . This quantitative PCR allowed to detect as few as 10 viral copies of the synthetic SARS-CoV-2 RNA control sample ( Figure 3B ).

For Nanopore DNA sequencing-based diagnostic, as described on Figure 2A , we have designed eight barcoded E-Sarbeco reverse primers (targeting the same amplicon region used for the quantitative PCR assay), and other eight barcoded E-Sarbeco forward primers (incorporating the required Gibson sequence), for concat-PCR amplification. This combinatorial strategy allows to mimic the screening of 64 pseudo-candidates ( Figure 3C ). Subsequent dilutions of the synthetic SARS-CoV-2 RNA control sample were used for reverse transcription, performed with defined barcoded E-Sarbeco primers (BC33-BC36;

BC38-BC40), in addition to a negative control sample devoid of viral RNA material (BC37) (dilution ID in Figure 3C ). The real-time diagnostic assay revealed significant differences between samples presenting different viral RNA copies based on the number of aligned read . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint counts towards the E-Sarbeco target region. Such differences, observed as early as 7 hours after DNA sequencing, revealed that samples issued from high viral RNA titers presented systematically higher amounts of aligned read counts to the E-Sarbeco target region ( Figure   3D and Figure S2 ). Furthermore, after 4 days of DNA sequencing (72h of DNA sequencing, and 1 day more for finalizing base-calling) a total of ~4 million reads were sequenced, corresponding to ~38 million monomers (9.7x concatemerization factor) ( Figure 3E) . Despite this large gain on monomer sequences, only 14 thousand optimal sequences (i.e. presenting both cDNA and PCR flaking barcodes and within the expected monomer amplicon insert size) were used for the diagnostic assay, all the others presenting either too short inserts (<100nt) or missing one or both of the required barcodes, essential for the identification of the samples. Noteworthy, the analysis of ~14 thousand optimal sequences was sufficient for discriminating samples with viral RNA titers ranging within 1 to 100 fold dilutions of the synthetic Covid-19 RNA material (Figure 3F & 3G) ; equivalent to 100 copies detected with the quantitative PCR assay ( Figure 3B) . Indeed, the sensitivity of the assay fails to discriminate in some cases between the negative control samples (BC37) and those issued from the least viral RNA titer (1/1000 dilution; BC36) ( Figure 3F & 3G) .

The few number of optimal sequences required for performing this diagnostic assay over 64 pseudo-candidates suggests that larger candidate screenings are possible within the scale of the sequencing-depth afforded by the MinION sequencer, anticipating the upscale of this strategy to several hundred of candidates without major impact on the sensitivity.

Considering the current needs on tracking SARS-CoV-2 variants' emergence and spreading over the population, we have evolved the aforementioned real-time SARS-CoV-2 diagnostic strategy into a variants' tracking system. Because all current described SARS-CoV-2 variants present an important number of mutations within the SPIKE gene, we have focused the realtime tracking system to this region. In fact, by concentrating the sequencing efforts towards . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint the SPIKE gene (~3.8kb size) we can afford to combine diagnostic and variants' tracking into a single assay, which per se represents a major progress relative to the current strategies.

In contrast to the aforementioned diagnostics effort targeting a short amplicon (E-Sarbeco), variants' tracking over the whole SPIKE gene uses a random-hexamer sequence for the reverse transcription step, and four primers targeting the SPIKE gene at different regions (~1kb distance among each primer) ( Figure 4A) . Like in the case of the diagnostics assay, the random-hexamer primer used for the reverse transcription present a unique molecular barcode as well as the common Gibson sequence. In contrary, the PCR primers (P1-P4) present a unique molecular barcode but they are devoid of the Gibson sequence. In fact, PCR amplification over cDNA issued from random-hexamer priming leads to amplicons of variable length (including fragments >1kb; see Figure 4B & Figure S3 ), thus the concatemerization step used on the short amplicon strategy does not appear essential. To simplify its use, PCR amplification is performed in presence of a pool of all four primers targeting the SPIKE region (P1-P4) and a Gibson primer sequence (in an equimolar concentration; i.e. 4x Gibson primer for 1x for each SPIKE targeting primer).

To better mimic a real situation, in which the diagnostics/variants' tracking assay is performed with samples collected from human candidates (e.g. nasopharyngeal swabs), human RNA -issued from cell lines in culture -has been combined with the synthetic viral RNA samples, such that the random-hexamer primers used for the reverse transcription assay could also provide means to count with a positive control for sample collection.

Furthermore, for addressing variants tracking performance, in addition to the Wuhan-Hu-1 SARS-CoV-2 RNA control sample, we have used synthetic viral RNA samples corresponding to the highly transmissible SARS-CoV-2 variant B.1.1.7, first described in the UK in December 2020, as well as the strain HF2393 described as a particular clade circulating in France since January 2020 9 ( Figure 4C ).

To perform diagnostics and variants' tracking assay over multiple pseudo-candidates, we have used eight barcoded random-hexamer primers for reverse transcription (BC37-BC44) in presence of different viral RNA titers (consecutive dilutions) and issued from the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint aforementioned synthetic viral RNA strains ( Figure 4D ). To multiply the number of pseudocandidates, we have amplified the eight barcoded cDNA products with four barcoded primers, either targeting the SPIKE gene (BC1-BC4 corresponding to pools of P1-P4) or targeting the E-Sarbeco region (BC5-BC8).

The cDNA products issued from barcoded-random-hexamer priming were first validated by quantitative PCR amplification, targeting either the human RNAse-P gene or the viral E-Sarbeco region ( Figure 4E) . Then, the PCR products generated with barcoded primers targeting SPIKE (BC1-BC4) and those targeting the E-Sarbeco region (BC5-BC8) were In addition to the diagnostic outcome, RETIVAD performs a variants' detection relative to the SARS-CoV-2 reference genome. In fact, as illustrated on Figure 4H , RETIVAD detected the presence of 7 significant variants within the SPIKE gene on all four samples issued from the BC37-barcoded cDNA (BC1-BC4 x BC37). Among them, five correspond to those previously described within the strain B.1.1.7 (N501Y, A570D, P681H, T716I and S982A); coherent with the fact that samples associated to the BC37 were generated with the synthetic viral RNA corresponding to such SARS-CoV-2 variant (Figure 4D) . Similarly, variants detection performed on samples issued from the BC40-barcoded cDNA (BC1-BC4 x BC40) revealed a similar signature, in despite of their lower coverage ( Figure 4I ). In fact, samples associated to the BC40 were also generated with the synthetic viral RNA corresponding to the B.1.1.7

strain, but with a 10-fold lower titer than in the case of the BC37 (Figure 4D) .

Finally, variants' tracking performed on samples issued from the BC38-barcoded cDNA (BC1-BC4 x BC38; French strain HF2393) revealed a single common mutation, D614G, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint previously described within the French strain HF2393 9 , but also among other European strains, including the B.1.1.7, as observed within the variants detected on samples issued from BC37 and BC40 (Figure 4H & I) .

Despite the current vaccination efforts, the pandemic situation anticipates the propagation of the recently described variants, as well as the emergence of new others, notably due to the major discrepancies between countries to access to optimal vaccination programs 6 . As consequence, counting with methodologies for Covid-19 diagnostics but also variants' tracking on a population scale remains essential.

Currently, diagnostics and variants' tracking are performed in two steps, which per se delays the time for obtaining a complete diagnostic. Furthermore, variants' tracking relies on full genome sequencing of SARS-CoV-2, which represent an expensive and labor-intensive effort, incompatible with population-scale assays. Finally, most of the variants' tracking efforts, relying on the use of large and expensive sequencing instruments, requires to centralize samples processing to dedicated institutions, which are rather absent on poor countries.

To address all these pitfalls, we propose herein a methodology able to (i) perform SARS-CoV-2 diagnostic and variants' tracking at the same time; (ii) shorten the time for accessing to the outcome of the diagnostic/variants' detection by the use of a real-time tracking system;

(iii) take advantage of the compact and affordable Oxford Nanoporer MinION DNA sequencing instruments, notably for deploying the aforementioned diagnostics and variants' tracking platform anywhere over the world.

The proposed variants' tracking focus on the analysis of the SPIKE gene instead of the whole SARS-CoV-2 genome. In fact, the SPIKE gene has shown to concentrate the majority of the currently discovered variants, which can be rationalized by the fact that it codes for the protein required for the interaction with the human cells. Noteworthy, most of the current vaccines against the Covid-19 are also based on the SPIKE region. Hence, concentrating . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint variants' tracking towards this gene appears an optimal compromise to increase the number of candidates to screen at once. This philosophy is shared by a recent work describing the HiSpike method, performing variants' detection within the SPIKE gene with the help of the small Illumina MiSeq instrument 10 . Illumina instruments being a short fragments sequencing approach, HiSpike requires 42 primer pairs generating amplicons of 400 nucleotides of length for covering the SPIKE region. In contrary, our proposed strategy, based on the use of long DNA fragments, compatible with the Nanopore DNA sequencing technology, relies on 4 primers targeting the SPIKE region, one random primer for the cDNA step and a common Gibson sequence. In fact, a strategy for SARS-CoV-2 diagnostic and variants' surveillance, dedicated to be deployed anywhere on the world does not only requires to count with low cost instruments, but also involve a reduced number of reagents for allowing its use on a long-standing manner.

For all these reasons, we are convinced that our proposed strategy, accompanied by the dedicated computational solution RETIVAD, can represent a game-changer approach for tracing variants' progression in the following months.

We gratefully acknowledge Corinne Cruaud and her team for the technical training on Oxford Nanopore sequencing. We also thank Veronique de Berardinis and Patrick Wincker for the logistics support required for this project.

This work was supported by DIM ELICIT's grants from Région Ile-de-France. 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The authors declare no competing interests.

Correspondence and requests for materials should be addressed to M.A.M.P. 

The following Synthetic SARS-CoV-2 RNA control samples, delivered at a concentration of 1 million copies per microliter has been used in this study:

-Wuhan-Hu-1 (GeneBankID: MN908947.3); Twist Biosc. ID: Ctrl2.

-France/HF2393/2020 (EPI_ISL_418227); Twist Biosc. ID: Ctrl7.

-England/205041766/2020 (EPI_ISL_710528); Twist Biosc. ID : Ctrl14 (B.1.1.7) .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Reverse transcription assays were performed using SuperScript™ IV Reverse Transcriptase (Figure 3B) .

Multiple reverse transcription assays -each of them performed in presence of a defined barcoded primer -were incubated at 50°C during 30 minutes, then at 80°C 10 minutes.

Barcoded cDNA samples were pooled (8 samples at once) and cleaned with SPRIselect reagent (Beckman; B23318) with a ratio of 0.5x. Cleaned cDNA is recovered on 80ul (half of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint the total initial cleaned volume; i.e. 2x concentration). 40ul of this pooled & cleaned cDNA will be used for PCR amplification (5ul x 8 PCR reactions; see below), while the remaining material is used for quantitative PCR evaluation.

Barcoded-cDNA material has been validated by quantitative PCR assay with the following primer sequences:

Primers targeting region on E gene of SARS-Cov-2 (annealing temp: 58°C: were pooled (4x2x25=200ul) and cleaned with SPRIselect reagent (Beckman; B23318) with a ratio of 0.5x.

Pooled & cleaned material is recovered on 50ul and used for Nanopore sequencing library preparation (Nanopore Ligation Sequencing Kit: SQK-LSK109). DNA libraries were sequenced on Nanopore FLO-MIN106D flowcells (R9) with the MinION Mk1C instrument (72h sequencing; high-accuracy base calling).

Real-time processing of the fastq files generated by the Mk1C instrument during sequencing is performed with our in-house developed RETIVAD (Real-Time Variants Detector) software.

RETIVAD collects each ten minutes the fastq files generated during sequencing from the Mk1C instrument and (i) search for Gibson sequences within the sequenced reads (regex query (https://pypi.org/project/regex) with maximum 4 mismatches accepted by default); (ii) split sequenced reads on monomers -delimited by the presence of the identified Gibson sequences; (iii) search for barcode sequences introduced during the reverse transcription (cDNA barcodes: BC33-BC48; regex query); (iv) search for barcodes introduced during the PCR amplification (PCR barcodes: BC1-BC8; regex query); (v) collect the sequences between the cDNA and PCR-associated barcodes for their alignment towards the SARS-CoV-2 reference genome (Wuhan-Hu-1 genome: NC_045512; BWA aligner following nanopore parameters). Considering that steps (iii) to (v) are performed within the retrieved monomers in step (ii), RETIVAD matches cDNA/PCR barcode combinations to aligned outcomes, thus providing a diagnostic outcome for the associated pseudo-candidate. Due to the low number of sequences retrieved at intervals of 10 minutes, and the relative short size of the SARS-CoV-2 genome (~29kb), RETIVAD is able to provide such diagnostic outcome within such 10 minutes interval. Hence, at the next round of data collection, RETIVAD reiterates on the aforementioned steps over the newly collected fastq files and appends the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.18.21257281 doi: medRxiv preprint outcome to the previous results. The continuous gain on aligned read-counts per region of interest (e.g. E-Sarbeco amplicon sequence, or SPIKE gene; defined as entry parameters) is visualized within scatterplot view (png file) updated during the processing (RETIVAD produces a png file per detected PCR barcode monomers; thus allowing to generate scatterplots displaying as many lines as detected cDNA barcode monomers; Figure S4 ).

In addition to the diagnostic outcome, RETIVAD has been designed for performing real-time variants detection. For it, the racon package (version 1.4.20) is used for raw de novo DNA assembly, followed by the alignment with Minimap2 (version 2.17). Medaka (version 1.2.5) is used to create a consensus sequence and variant calls from the aligned data (> 20 aligned reads required as default for variants calling). RETIVAD generates BAM files per cDNA/PCR barcode combination harboring base conversion information and their associated coverage (for visualization with Genome browsers like IGV). Furthermore, summary files per cDNA/PCR barcode combination are also generated, providing the genomic position in which the base conversion has been detected, the identity of the converted bases as well as their associated coverage. Like in the case of the diagnostics processing, variants detection is performed in real time, thus providing the possibility to the user to visualize the results and decide whether sequencing requires still to be performed or whether the collected information is sufficient to conclude the diagnostics/variants tracing for the corresponding candidates.

RETIVAD generates a final summary report for the diagnostics outcome when no new fastq files are available after 40 minutes from the last data collection and processing.

All genome sequences included in this study are available under request.

RETIVAD is freely available at https://github.com/SysFate/retivad 

Considerations for diagnostic COVID-19 tests

International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted

Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays

REMBRANDT: A highthroughput barcoded sequencing approach for COVID-19 screening

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding

Swab-Seq: A high-throughput platform for massively scaled up SARS-CoV-2 testing

Understanding variants of SARS-CoV-2

Hospitalization and mortality associated with SARS-CoV-2 viral clades in COVID-19

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Introductions and early spread of SARS-CoV-2 in France

HiSpike: A high-throughput cost effective sequencing method for the SARS-CoV-2 spike gene

International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted