PONE-D-23-41807NGS method for parallel processing of high quality, damaged or fragmented input material using target enrichmentPLOS ONE

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When responding to reviewer comments, please make sure to clearly elaborate/explain differences in results from FFPE1-4 and Horizon reference samples. Also, differentiate your research article from a lab protocol, based on your provided data.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. 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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. 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: Lyander et al. “NGS method for parallel processing of high quality, damaged or fragmented input

material using target enrichment”

In this article, the authors describe a procedure for NGS library preparation form different types of samples.

I have the following questions/comments:

1. Were the same input amounts used for FFPE1-4 and the Horizon reference samples?

2. What QC metrics are used for the 2 panels described by the authors? The reported duplication rates, target coverage and %target bases for the FFPE samples would, in many clinical laboratories, be considered as failures or not passing QC.

3. Can the authors comment on the differences in QC metrics in Figure 3 and 4, which are using different samples and panels. The results seem better with the Horizon reference samples and panel 2. Do the authors think this is due to the difference in panels/probes, or due to a difference between “real” samples and commercial reference samples?

4. Table 4. Can the authors also provide the quality score for the variant calls?

5. Table 4. Can the authors include data for HD777 in the table.

6. Can the authors also include the total number of variants detected for the samples? In many cases, detecting the expected variants is not the problem with compromised samples, but instead with high duplication rates, early PCR errors result in a high number of artifactual calls and a large number of false positive variants.

Reviewer #2: The manuscript entitled "NGS method for parallel processing of high quality, damaged or fragmented input

material using target enrichment" described a joint procedure for preparing enriched

DNA libraries from high molecular weight DNA and DNA from formalin-fixed, paraffinembedded tissue, fresh frozen tissue material, as well as cell-free DNA.

- The Authors should provide the expand forms for all acronyms, including gene acronyms, through the text when they first appear.

- Gene acronyms should be written in italics.

Reviewer #3: The sutdy was conducted in a very relevant topic. The author very elaborately detailed an alterantive in a challenging issuee. I onlye suggest if author provide a pictorial diagram outlining the steps for the workflow, highlighting important QC steps which one should be aware of, will be helpful for the readers.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Subit Barua

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NGS method for parallel processing of high quality, damaged or fragmented input material using target enrichment

PONE-D-23-41807R1

Dear Dr. Anna,

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.

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Kind regards,

Elingarami Sauli, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

The decision to accept this submission was reached based on  the fact that, the authors  have responded on a previous query regarding the input amounts for FFPE1-4 and the Horizon reference used for the samples, which you have added to the Methods section, in “Enzymatic fragmentation of gDNA”.

As regards to raised query on QC metrics used for the 2 panels, as described on duplication rates, target coverage and %target bases for the FFPE samples, which would in many clinical laboratories be considered as failures or not passing QC, the authors have agreed with this and indicated that, in order to improve the QC metrics, a larger amount of DNA from the FFPE material could be used. In addition, the authors affirm that, deeper sequencing can improve the QC metrics. The authors have further clarified that, samples used in this case were of poor quality DNA intended to show how data could be improved with an alternative laboratory protocol, and may not be fully representative for all clinical cases.

As regards to a query on differences in QC metrics in Figure 3 and 4, which are using different samples and panels. The results seem better with the Horizon reference samples and panel 2. The authors have agreed that, yes the results seem better with the Horizon reference samples compared to the FFPE samples, which is possibly/likely due to the difference in quality between the commercial formalin-comprised Horizon samples (not FFPE material) HD798, HD799 and HD803 with a DIN value of 7.3, 3.9 and 1.7, respectively, and the formalin fixed paraffin embedded patient samples with DIN 1.0, 1.1, 1.0, 1.7.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. 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 #2: Yes

Reviewer #4: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #4: N/A

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4. 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 #2: Yes

Reviewer #4: Yes

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5. 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 #2: Yes

Reviewer #4: Yes

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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 #2: The Authors have addressed all my concerns and I have no further comments.

The manuscript is suitable for publication.

Reviewer #4: Developing a protocol that is optimized for all sample types is relevant to clinical practice. Therefore, testing real world clinical samples (as many as possible) to demonstrate that the protocol is robust with accuracy, reproducibility and limits of detections acceptable for clinical practice is desired.

1. It remains not entirely clear to me if cf DNA has been subjected to enzymatic fragmentation in the combination protocol. In Figure 1, it states that “the first step is enzymatic fragmentation, which is performed for all samples except cfDNA samples, and the following end-repair/A-tailing step is a combination of KAPA HyperPrep and HyperPlus protocols (hereafter denoted combination protocol)”. However, in page 7, line 149, under the section of enzymatic fragments of gDNA, HD777, a cf DNA was included in the experiment. Confusedly, in page 5 under the section of Library preparation, it states that “for a combined preparation of both gDNA and cfDNA, the Hyperplus kit (includes enzymatic fragmentation) is used. However, if there solely cfDNA samples, the HyperPre kit (without enzymatic fragmentation) should be used instead”. Please make consistent statements throughout the manuscript and clearly indicate if one or two kits are required for the proposed combination protocol.

2. Table 2. Could the authors be more specific about the selection of alternative 1 or alternative 2? The selection based on “majority” of samples is vague and imprecise. Is 5 out of 8 or 7 out of 8 considered “majority”?

3. My main concern is that the combination protocol may introduce false positive variant/artificial calls or false negative results in the FFPE samples due to over-treatment. For example in Table 4, NRAS: Q61K with VAF of 12% was not detected in one of the samples but required manual inspection of IGV to find the variant. I suggest the authors to evaluate more clinical FFPE samples with known variants detected by orthogonal methods. Presenting the performance data of applying combination protocol on FFPE samples including sensitivity, specificity, reproducibility and percent of failure rate could significantly strengthen the conclusions of this manuscript.

4. Using cfDNA sample with 5% VAF is not ideal to evaluate the performance of a protocol designing for clinical practice. Clinically, the lower limit of detection of cfDNA is in general set at 0.1% VAF. Including cfDNA samples with lower VAFs for evaluation is highly recommended.

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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 #2: No

Reviewer #4: No

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