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. 2023 Sep 14;18(9):e0288687. doi: 10.1371/journal.pone.0288687

A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay

Jason D Limberis 1,*, Alina Nalyvayko 1, Joel D Ernst 1, John Z Metcalfe 2
Editor: Padmapriya P Banada3
PMCID: PMC10501585  PMID: 37708184

Abstract

Targeted amplicon sequencing to identify pathogens, resistance-conferring mutations, and strain types is an important tool in diagnosing and treating infections. However, due to the short read limitations of Illumina sequencing, many applications require the splitting of limited clinical samples between two reactions. Here, we outline hairpin Illumina single-tube sequencing PCR (hissPCR) which allows for the generation of overlapping amplicons containing Illumina indexes and adapters in a single tube, effectively extending the Illumina read length while maintaining reagent and sample input requirements.

Introduction

Targeted amplicon sequencing enables the identification of resistance-conferring mutations and strain typing of important human pathogens. However, due to the short read limitations of Illumina sequencing–a maximum of 600 base pairs (bp) when 300bp paired-end sequencing is done–regions larger than 600bp require overlapping amplicons that necessitate splitting a sample between two reactions [1]. However, clinical samples are often limited, and nucleic acid concentrations are difficult to quantify, increasing the chance of sequencing failure. For example, DNA extraction of Mycobacterium tuberculosis, the etiological agent of tuberculosis, is most commonly done from patient sputum. This results in the co-extraction of human DNA including oral and respiratory flora, thus complicating the quantification of M. tuberculosis DNA in a sample. Furthermore, with as few as 5,000 bacteria/ml in the sputum of a culture positive patient [2], there is limited material to work with. Additionally, standard library preparation is complex [3] and requires numerous enzymatic reactions leading to multiple vulnerabilities within targeted amplicon sequencing workflows and complicating point-of-care or near point-of-care applications.

Here, we outline a hairpin Illumina single-tube sequencing PCR (hissPCR), a method that allows for the generation of sequenceable, overlapping amplicons in a single tube, effectively tripling Illumina read length. hissPCR uses target-specific primers with universal tail sequences. In the first round of PCR, primers containing a universal tail sequence amplify a target up to 1200 nucleotides (Fig 1, step 1). In the second round of PCR, tailed gene-specific primers target the amplicon generated in step 1. Together with the universal tailed primers containing the index and adapter sequence, generate two overlapping amplicons of sequenceable length (Fig 1, step 2). In a traditional PCR, the overlapping primers (F2 and R2) would amplify not only the original template but also subsequent amplicons, overtaking the reaction with amplicons ~100 nucleotides in length. In hissPCR however, the gene-specific primers (F2 and R2) contain the same tailed sequence. These tails form a hairpin during amplification (Fig 1, step 2), nullifying the formation of the 100 base pair overlap amplicon and allowing amplification of the intended ~600 base pair amplicons. Paired-end sequencing (Fig 1, step 3) then generates the full 1200 base pair sequence. Several of these primers can be designed to overlap, covering even larger areas.

Fig 1. Hairpin Illumina single-tube sequencing PCR (hissPCR).

Fig 1

Open in a new tab

In step 1, primers containing a universal tail sequence amplify a target up to 1200 nucleotides flanking each gene of interest. In step 2, tailed gene-specific primers target the amplicon generated in step 1, and, together with the universal tailed primers that contain the index and adapter sequence, generate two overlapping amplicons of sequenceable length while the gene-specific primers (Forward-1 [F1] and Reverse-2 [R2], each containing the same tailed sequence), form a hairpin during amplification, thus nullifying the formation of the 100 base pair overlap amplicon and allowing amplification of the intended ~600 base pair amplicons.

Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io, DOI 10.17504/protocols.io.q26g7yw79gwz/v1, and is included for printing as S1 File with this article.

Expected results

There will be several amplicons for each target (Fig 2). The amplicons will include the large amplicon, and the two small amplicons, with no Illumina adapters, an Illumina adapter on one end of the amplicon, or Illumina adapters on both ends of the amplicon (the sequenceable amplicons). The large amplicons are not sequenceable as they have the same adapter at both ends, even if it is generated in the second PCR using the Illumina adapter primers. The qPCR ratio of the Illumina inner and outer primer sets will provide the proportion of DNA in the sample with Illumina adapters on both ends of the amplicon (the sequenceable amplicons), while the melt curve will provide information on the proportion of each amplicon in the sample. Alternatively, amplicons can be quantified using commercial Illumina library quantification kits.

Fig 2. Expected results.

Fig 2

Open in a new tab

A) hissPCR generated amplicons using two forward and two reverse overlapping primers in a single tube. Amplicons contain either no Illumina adapter, one Illumina adapter, or two Illumina adapters per amplicon. B) The relative proportion of amplicons in the sample containing both Illumina adapters. C) Melt curves showing the expected amplicons for each primer set and the ratio of amplicons containing both Illumina adapters.

Supporting information

S1 File. Step-by-step protocol, also available on protocols.io, DOI 10.17504/protocols.io.q26g7yw79gwz/v1.

(PDF)

Click here for additional data file. (699.4KB, pdf)
S2 File. Raw output from D1000 Agilent TapeStation of hissPCR generated amplicons using two forward and two reverse overlapping primers in a single tube as shown in Fig 2A.

(CSV)

Click here for additional data file. (13.2KB, csv)
S3 File

(PDF)

Click here for additional data file. (411.5KB, pdf)
S1 Raw images

(PDF)

Click here for additional data file. (89.4KB, pdf)

Data Availability

All relevant data are within the paper, its Supporting information files, and the Github hssPCR repository https://github.com/SemiQuant/hissPCR.

Funding Statement

JZM, 1R01AI153213, National Institute of Allergy and Infectious Diseases (NIAID), https://www.niaid.nih.gov/, The funders did not and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

Decision Letter 0

Padmapriya P Banada

23 Mar 2023

PONE-D-23-05939A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay.PLOS ONE

Dear Dr. Limberis,

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.

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Padmapriya P Banada, PhD

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

Thank you for submitting your protocol and I hope the comments from the reviewers would help improve the methodology and increased success of the proposal. In general, I agree with the reviewers that the protocol limitations and potential solutions should be clearly mentioned. Although reviewer 1 has major concerns on the amplicon size and illumina limitations, please expand on this clearly in your introduction.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

4. If the manuscript contains new data, have the authors made this data 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: No

Reviewer #2: N/A

Reviewer #3: No

**********

5. Is the article 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 highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay, suggests novel approach for generating longer amplicons using illumina sequencing. Their approach uses a hairpin Illumina single-tube sequencing PCR (hissPCR), a method that allows for the generation of sequenceable, overlapping amplicons in a single tube, effectively tripling Illumina read length.

Although their approach aims to address a critical issue in the field, they do not provide any proof that their approach works. They mention in their intro that with 300bp paired end sequencing, they can sequence an amplicon that is 600bp long. However, paired end sequencing is designed to produce high quality coverage for the region being sequenced (300bp) not 600bp. Furthermore, 300bp paired end sequencing is limited to certain sequencing instruments which should be mentioned in their intro and methods.

Furthermore, illumina sequencing was designed to be accurate for short reads and the authors do not explain how they plan to overcome issues in accuracy with longer reads or how they plan to even sequence longer reads (single read vs paired reads, specific instruments, library prep, etc). They also do not provide any information on expected read coverage and how those reads could be potentially used for downstream analysis particularly for TB clinical assays.

Reviewer #2: Dear authors, I have following suggestions to improve the manuscript:

1) Figure 1: Step 2 please align the R1 primers in the F2R1 amplicon with the R1 primers in the F1R1 amplicon. It looks misleading as it is.

2) Figure 1: Step 3: please correct spelling of "generation".

3) Methods and material: Please describe in detail target identification and primer design for a single-plex reaction sequenceable overlapping amplicons, to ensure the hairpin structure of short 100bp amplicon is retained.

4) Expected results:

a. Please describe in detail any short comings of this method, essential precautions partaken to ensure minimum contamination and best output, including but not limited to QC.

b. Please include comments on the bioinformatics pipeline and computational methods used for clean up of the data.

c. Finally, please also include comments on whether single -plex reaction has impact on improved efficiency, coverage and cost of the sequencing.

Reviewer #3: Manuscript present an new PCR assay to amply longer genomic intervals in microbial genome using a nested PCR strategy. The approach is not entirely novel but protocol developed by authors does makes sense and is described appropriately. I have following comments and questions for authors:

1. Given that amplified product contains fragments of multiple sizes, precise quantification of library before sequencing may be challenging and would need accurate nanomole calculation.

2. Authors have not shown how efficiently they were able to clean the longer amplicons using bead ratio? A tapestation image of library after the bead clean up would be helpful. A band of high molecular weight is evident in the amplicon lane.

3. Accurate library quantification before loading the flow cell is crucial for optimal cluster generation on target. It may cause under or over clustering and failure of sequencing run!

4. Would assay perform similarly in genomes with different GC ratio and intervals with more repetitive sequences or structural variants? Any thoughts?

5. Direct whole genome sequencing using long-read sequencing method is feasible now. What authors think of those options to address this question instead of classical nested PCR approach.

6. Would time and reagent costs in presented protocol vs whole genome sequencing approach, would be significantly different? Any thoughts

7. Manuscript does not provide any data generated using this protocol. How did pathogen identification, taxonomic classification and mutation detection analysis go ? It is important to validate the intended application of this protocol?

Overall, manuscript does present a new protocol to amplify targeted regions in the pathogen genome. If authors agree to address raised points, I would recommend publication of this manuscript.

**********

7. 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.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Prithvi Raj, Ph.D. Microbiome Research Lab, UT Southwestern Medical Center, Dallas, Texas, USA

**********

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PLoS One. 2023 Sep 14;18(9):e0288687. doi: 10.1371/journal.pone.0288687.r002

Author response to Decision Letter 0

16 May 2023

PONE-D-23-05939

A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay.

PLOS ONE

Additional Editor Comments (if provided):

Thank you for submitting your protocol and I hope the comments from the reviewers would help improve the methodology and increased success of the proposal. In general, I agree with the reviewers that the protocol limitations and potential solutions should be clearly mentioned. Although reviewer 1 has major concerns on the amplicon size and illumina limitations, please expand on this clearly in your introduction.

Thank you for the opportunity to submit a revised protocol, and we appreciate the reviewers' comments and suggestions. We have carefully considered each comment and made the necessary revisions to strengthen the manuscript. Reviewer 1 raised concerns about the amplicon size and Illumina limitations. We have expanded the introduction to provide a clear explanation and have included supporting sequencing data to demonstrate the success of the method. Reviewer 2 suggested adding more detail on the sample analysis process and Reviewer 3 recommended including accurate library quantification all of which are now included in the manuscript. We have responded to the specific concerns below (in blue text) and made the relevant changes to the manuscript and protocol. These revisions have significantly improved the quality of the manuscript, and we hope that you and the reviewers find them satisfactory. Thank you for your consideration, and we look forward to hearing from you.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Yes

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

4. If the manuscript contains new data, have the authors made this data 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: No

Reviewer #2: N/A

Reviewer #3: No

5. Is the article 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 highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay, suggests novel approach for generating longer amplicons using illumina sequencing. Their approach uses a hairpin Illumina single-tube sequencing PCR (hissPCR), a method that allows for the generation of sequenceable, overlapping amplicons in a single tube, effectively tripling Illumina read length.

Although their approach aims to address a critical issue in the field, they do not provide any proof that their approach works. They mention in their intro that with 300bp paired end sequencing, they can sequence an amplicon that is 600bp long. However, paired end sequencing is designed to produce high quality coverage for the region being sequenced (300bp) not 600bp. Furthermore, 300bp paired end sequencing is limited to certain sequencing instruments which should be mentioned in their intro and methods.

Thank you for your concerns. We have added supporting sequencing data to demonstrate the success of the method. The output of the alignments is now in Figure 2. Regarding the sequencing length, when performing pair-end sequencing, each read in the pair is sequenced the number of cycles programmed (i.e., for 250bp paired end sequencing, a 500bp region of the individual DNA, 250bp from each end). Thus, if an amplicon of 500bp is sequenced, the entire 500bp will be reported; when 300bp paired end sequencing is used, the maximum coverage of a single amplicon is 600bp.

Line 19: Targeted amplicon sequencing enables the identification of resistance-conferring mutations and strain typing of important human pathogens. However, due to the short read limitations of Illumina sequencing – a maximum of 600 base pairs (bp) when 300bp paired-end sequencing is done – regions larger than 600bp require overlapping amplicons that necessitate splitting a sample between two reactions [1].

Line 45: Three hundred bp paired-end sequencing (Figure 1, step 3) then generates the full 1200 base pair sequence (an amplicon of up to four times the sequencing length can be sequenced per reaction, e.g., 4 x 250bp paired-end sequencing = 1000bp). Several of these primers can be designed to overlap, covering even larger areas.

Line 90: D) An output from the hissPCR analysis pipeline showing the expected read coverage over the target area using 250bp Illumina sequencing.

Furthermore, illumina sequencing was designed to be accurate for short reads and the authors do not explain how they plan to overcome issues in accuracy with longer reads or how they plan to even sequence longer reads (single read vs paired reads, specific instruments, library prep, etc).

Illumina sequencing is highly accurate but has limited read length (common kits allow for 50, 75, 150, 250, and 300bp sequencing reactions). However, the length of the DNA sequenced does not adversely affect the process or quality (https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000003874), and several Illumina PCR amplicon-based kits sequence short (275bp) PCR products (AmpliSeq product line, https://www.illumina.com/products/by-brand/ampliseq.html). It is also possible, using paired end sequencing, to sequence the same portion of DNA from both directions (reads) when the fragment is smaller than the read length (Figure below). This is used in some cancer assays to increase confidence in the base calls and allow for the detection of minor heterogeneous populations as is common in cancer samples.

They also do not provide any information on expected read coverage and how those reads could be potentially used for downstream analysis particularly for TB clinical assays.

This is determined by the end-user; however, we have added some guidelines to the protocol (“Depth and Pooling Calculations”) detailing how to accurately calculate the library for optimal flow cell loading and sequencing.

Reviewer #2: Dear authors, I have following suggestions to improve the manuscript:

1) Figure 1: Step 2 please align the R1 primers in the F2R1 amplicon with the R1 primers in the F1R1 amplicon. It looks misleading as it is.

2) Figure 1: Step 3: please correct spelling of "generation".

3) Methods and material: Please describe in detail target identification and primer design for a single-plex reaction sequenceable overlapping amplicons, to ensure the hairpin structure of short 100bp amplicon is retained.

Thank you for your comments, we have edited Figure 1 for clarity and incorporated your suggestions. We have also added a section to the protocol (“Primer design”) that details the primer design process and have published a pipeline on GitHub to assist users with their design (https://github.com/SemiQuant/hissPCR).

4) Expected results:

a. Please describe in detail any short comings of this method, essential precautions partaken to ensure minimum contamination and best output, including but not limited to QC.

We have added the shortcomings to the manuscript: the main limitation is the possibility of uneven amplicon ratios in the sample, which we specifically address in the manuscript.

Line 70: “While hissPCR should not adversely affect Illumina cluster generation and sequencing, its is important to accurately quantify the sequenceable material to load the appropriate amount on the flow cell; however, since not all the amplicons will be present at the same ratios, we recommend aiming for a 1.2-1.5x higher than the desired coverage (this will be especially important for multiplexed hissPCR reactions).”

b. Please include comments on the bioinformatics pipeline and computational methods used for clean up of the data.

We have added a section to the protocol (“Data Analysis”) that details the methodology to analyze the Illumina sequencing data produces. We have also published a pipeline on GitHub to assist users with their analyses.

Line 73: The sequence data can be analyzed using most amplicon processing pipelines; however, the primer sequences must be trimmed from the reads to eliminate their effect on mutation identification. Our analysis pipeline utilizes the `samtools ampliconclip` command to remove these.

c. Finally, please also include comments on whether single -plex reaction has impact on improved efficiency, coverage and cost of the sequencing.

We have included a comment on how hissPCR does not affect the efficiency of sequencing. However, coverage and cost of sequencing are subject to numerous factors, which will differ depending on how the user utilizes the method.

Reviewer #3: Manuscript present an new PCR assay to amply longer genomic intervals in microbial genome using a nested PCR strategy. The approach is not entirely novel but protocol developed by authors does makes sense and is described appropriately. I have following comments and questions for authors:

1. Given that amplified product contains fragments of multiple sizes, precise quantification of library before sequencing may be challenging and would need accurate nanomole calculation.

2. Authors have not shown how efficiently they were able to clean the longer amplicons using bead ratio? A tapestation image of library after the bead clean up would be helpful. A band of high molecular weight is evident in the amplicon lane.

3. Accurate library quantification before loading the flow cell is crucial for optimal cluster generation on target. It may cause under or over clustering and failure of sequencing run!

Responses 1-3: Thank you for these comments and suggestions. We have added in a section to the protocol (“Depth and Pooling Calculations”) detailing how to accurately calculate the library for optimal flow cell loading and sequencing. The figure in the protocol shows the amplicons post bead cleanup, while these do still contain non-sequenceable material, they will not affect the Illumina cluster generation as they fail to bind to the flowcell.

4. Would assay perform similarly in genomes with different GC ratio and intervals with more repetitive sequences or structural variants? Any thoughts?

We expect hissPCR to perform as a regular PCR if the annealing temperature is >55C, regardless of the GC content. The hairpin is formed predominantly by the tail sequences which are constant, so structural variants should also perform like regular end-point PCR.

5. Direct whole genome sequencing using long-read sequencing method is feasible now. What authors think of those options to address this question instead of classical nested PCR approach.

While whole genome sequencing using long-read sequencing is feasible, it requires a large input of DNA which is not present in all clinical samples and needs more sequencing reads than targeted sequencing. Furthermore, in many clinical samples (e.g., sputa), the majority of the DNA is from the host rather than the pathogen of interest, rendering direct whole genome sequencing difficult.

6. Would time and reagent costs in presented protocol vs whole genome sequencing approach, would be significantly different? Any thoughts

The reagent cost would be less than regular, split sample PCR, and on the order of what whole genome sequencing approaches. However, this difference will depend on numerous factors chosen by the user, including sequencing depth, genome and target size, and the number of samples available for pooling.

7. Manuscript does not provide any data generated using this protocol. How did pathogen identification, taxonomic classification and mutation detection analysis go? It is important to validate the intended application of this protocol?

We have included sequencing results now in Figure 2 (panel D) and found base calls the same as end-point PCR.

Overall, manuscript does present a new protocol to amplify targeted regions in the pathogen genome. If authors agree to address raised points, I would recommend publication of this manuscript.

7. 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.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Prithvi Raj, Ph.D. Microbiome Research Lab, UT Southwestern Medical Center, Dallas, Texas, USA

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. 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.

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Decision Letter 1

Padmapriya P Banada

2 Jul 2023

A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay.

PONE-D-23-05939R1

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Thank you for resubmitting your manuscript and considering reviewers comments positively. I do see that the methodology described is more accurate, clear and understandable. I accept that rebuttal provided for all the reviewers comments.

Reviewers' comments:

Acceptance letter

Padmapriya P Banada

5 Sep 2023

PONE-D-23-05939R1

A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay.

Dear Dr. Limberis:

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.

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on behalf of

Dr. Padmapriya P Banada

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File. Step-by-step protocol, also available on protocols.io, DOI 10.17504/protocols.io.q26g7yw79gwz/v1.

    (PDF)

    Click here for additional data file. (699.4KB, pdf)
    S2 File. Raw output from D1000 Agilent TapeStation of hissPCR generated amplicons using two forward and two reverse overlapping primers in a single tube as shown in Fig 2A.

    (CSV)

    Click here for additional data file. (13.2KB, csv)
    S3 File

    (PDF)

    Click here for additional data file. (411.5KB, pdf)
    S1 Raw images

    (PDF)

    Click here for additional data file. (89.4KB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (935.9KB, docx)

    Data Availability Statement

    All relevant data are within the paper, its Supporting information files, and the Github hssPCR repository https://github.com/SemiQuant/hissPCR.

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