Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR

Diyanath Ranasinghe; Tibutius Thanesh Pramanayagam Jayadas; Deshni Jayathilaka; Chandima Jeewandara; Osanda Dissanayake; Dinuka Guruge; Dinuka Ariyaratne; Dumni Gunasinghe; Laksiri Gomes; Ayesha Wijesinghe; Ruwan Wijayamuni; Gathsaurie Neelika Malavige

doi:10.1371/journal.pone.0265220

Peer Review History

Original SubmissionNovember 30, 2021
10 Jan 2022 Decision Letter - Ruslan Kalendar, Editor PONE-D-21-37979Comparison of different sequencing techniques with multiplex real-time PCR for detection to identify SARS-CoV-2 variants of concernPLOS ONE Dear Dr. Malavige, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Feb 24 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Reviewer #1: In this study, RTPCR for single nucleotide polymorphisms was high accuracy for VOC identification. There were three false positive results for B.1.617 due to an isolated L452R mutation but B.1.1.7 or B.1.351 calls were 100% accurate. The authors also compared Oxford Nanopore technology (ONT) to Illumina sequencing. Higher call accuracy was observed with Illumina among 37 samples consistent with what would be expected but ONT had higher sequence coverage. This study was conducted prior to emergence of the omicron VOC and further experiments would need to be performed to evaluate these RTPCRs for differentiation from omicron. Overall, this was a proof-of-concept study that RTPCR may be useful for differentiating known predominant VOCs and ONT is acceptable in resource-limited settings. This is a relevant topic but there were major methodological limitations. Major limitations: While ONT and Illumina Nextseq 550 platforms are compared, they are then used interchangeably for the RTPCR comparison. The results should be separated for comparisons to each gold standards. The immediate translation of RTPCR findings is limited by the shift to the omicron variant, but there is still utility in this study as a proof-of-concept. If the accuracy was unchanged between sequencing platforms, this needs to be described. This is particularly relevant when 7 of 27 lineage calls were different by different methods. A table for information about the RTPCR performance compared to each sequencing method would be helpful to this end. The authors should consider including a 2x2 table for sensitivity/specificity in the supplement or describe it more clearly (i.e., the % agreement) in the results to support statements of good sensitivity and accuracy. The dates or months that samples were collected would be useful to put into context each sequencing method for the RTPCR comparison. The dates/months that variants were introduced in this population should also be described. Minor: The title "Comparison of different sequencing techniques with multiplex real-time PCR for detection to identify SARS-CoV-2 variants of concern" is not grammatically correct. It should be "for detection" or "for identification" In the abstract, regarding the sentence “34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using the Illumina generated <15% ambiguous bases”, it is not clear why cutoffs change. The authors could use the sentence from the results “samples with less than 10% ambiguous bases were higher for ONT compared to 21 Illumina (29 vs 25).” Only two RTPCR panels described. In abstract and the introduction, the authors should state two panels by the same commercial vendor are used. Currently, it seems like many panels were going to be compared. Under methods, it is unclear what kit, primers or probes were used for initial detection for the 30 cycle threshold cut off. It should be described if RTPCR was run in duplicate or triplicate. “Due to improved coverage over the SARS-CoV-2 genome, 1 more samples which were sequenced using the ONT platform were lineage call-able by both Pango and Scorpio nomenclature.” – Please list the number in the text and/or cite the table. In the discussion, I would recommend writing “less expensive” rather than “cheaper” as a potential benefit. Cost effectiveness was not evaluated. So, I would consider reframing the language to state that the error rate is acceptable when resources for Illumina sequencing may not be available. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. 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If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0265220.r001
Revision 1
12 Feb 2022 Author Response 7th February 2022 Dr. Ruslan Kalendar Academic Editor PLOS ONE Dear Dr. Kalendar, Submission of manuscript titled ‘Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR’ We wish to thank the reviewers for carefully going through our manuscript and the useful suggestions they have made. We have incorporated all these changes in the revised version and letter and have addressed all issues raised by the reviewers. We have highlighted the changes in the revised manuscript. Reviewer #1: In this study, RTPCR for single nucleotide polymorphisms was high accuracy for VOC identification. There were three false positive results for B.1.617 due to an isolated L452R mutation but B.1.1.7 or B.1.351 calls were 100% accurate. The authors also compared Oxford Nanopore technology (ONT) to Illumina sequencing. Higher call accuracy was observed with Illumina among 37 samples consistent with what would be expected but ONT had higher sequence coverage. This study was conducted prior to emergence of the omicron VOC and further experiments would need to be performed to evaluate these RTPCRs for differentiation from omicron. Overall, this was a proof-of-concept study that RTPCR may be useful for differentiating known predominant VOCs and ONT is acceptable in resource-limited settings. This is a relevant topic but there were major methodological limitations. Major limitations: 1) While ONT and Illumina Nextseq 550 platforms are compared, they are then used interchangeably for the RTPCR comparison. The results should be separated for comparisons to each gold standards. The immediate translation of RTPCR findings is limited by the shift to the omicron variant, but there is still utility in this study as a proof-of-concept. Response: We highly value and appreciate the reviewer’s suggestions. We have separated the RT-PCR vs NGS comparison into two sections calculating percentages for each NGS technology, in the revised version of the manuscript. We also completely agree with the comment the reviewer has made regarding detection of Omicron. Since the spread of the Omicron variant in Sri Lanka, we are in the process of assessing our in-house RTPCR assay for identifying Omicron and we have been able to successfully differente Omicron BA.1 from other variants (by presence of E484K, N501Y, HV69/70, L452R, and K417N). We have also been able to identify BA.2 from BA.1 due to the absence of HV69.70 in BA.2. However, as we have just started assessing the usefulness of this RTPCR for BA.1 and BA.2, we have not included this data in this manuscript. 2) If the accuracy was unchanged between sequencing platforms, this needs to be described. This is particularly relevant when 7 of 27 lineage calls were different by different methods. Response: We thank the reviewer for this suggestion. This section was rewritten taking genome coverage into account and ignoring the differences in sub lineages (AY classification was changed by Pango team after we submitted the paper so it can be confusing to use those obsolete sub lineages). Only a classification at VOC level was used (pango lineage version 2021-08-09) 3) A table for information about the RTPCR performance compared to each sequencing method would be helpful to this end. The authors should consider including a 2x2 table for sensitivity/specificity in the supplement or describe it more clearly (i.e., the % agreement) in the results to support statements of good sensitivity and accuracy. Response: we thank the reviewer for this suggestion. We have added summary table and a Venn diagram to supplementary table 2. 4) The dates or months that samples were collected would be useful to put into context each sequencing method for the RTPCR comparison. The dates/months that variants were introduced in this population should also be described. Response: We thank the reviewer for this suggestion. We have included the collection dates and deposited dates for all the samples in supplementary table 1. In addition, we have added a few lines to the methods section regarding the outbreak periods. Minor: 5) The title "Comparison of different sequencing techniques with multiplex real-time PCR for detection to identify SARS-CoV-2 variants of concern" is not grammatically correct. It should be "for detection" or "for identification" Response: We apologize for this mistake and we have corrected it as suggested by the reviewer. 6) In the abstract, regarding the sentence “34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using the Illumina generated <15% ambiguous bases”, it is not clear why cutoffs change. The authors could use the sentence from the results “samples with less than 10% ambiguous bases were higher for ONT compared to 21 Illumina (29 vs 25).” Response: We thank the reviewer for pointing this out. We have corrected this throughout the text. 7) Only two RTPCR panels described. In abstract and the introduction, the authors should state two panels by the same commercial vendor are used. Currently, it seems like many panels were going to be compared. Response: We apologize for this mistake. We have corrected this in the revised version of the manuscript. 8) Under methods, it is unclear what kit, primers or probes were used for initial detection for the 30-cycle threshold cut off. It should be described if RTPCR was run in duplicate or triplicate. Response: We apologize for this omission. We have included this in the methods section in the revised version. . 9) “Due to improved coverage over the SARS-CoV-2 genome, 1 more sample which were sequenced using the ONT platform were lineage call-able by both Pango and Scorpio nomenclature.” – Please list the number in the text and/or cite the table. Response: we thank the reviewer for pointing out this omission. We have corrected this in the revised version. 10) In the discussion, I would recommend writing “less expensive” rather than “cheaper” as a potential benefit. Cost effectiveness was not evaluated. So, I would consider reframing the language to state that the error rate is acceptable when resources for Illumina sequencing may not be available. Response: We agree with the reviewer that this is a more suitable word to use. We have corrected this in the revised version of the manuscript. Thank you for considering our manuscript for publication with PLOS One. Yours Sincerely, Prof. Neelika Malavige Attachments Attachment Submitted filename: Response to Reviewers_1.docx https://doi.org/10.1371/journal.pone.0265220.r002
28 Feb 2022 Decision Letter - Ruslan Kalendar, Editor Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR PONE-D-21-37979R1 Dear Dr. Malavige, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ruslan Kalendar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265220.r003
Formally Accepted
15 Mar 2022 Acceptance Letter - Ruslan Kalendar, Editor PONE-D-21-37979R1 Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR Dear Dr. Malavige: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Ruslan Kalendar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265220.r004

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