Evaluation of the correctable decoding sequencing as a new powerful strategy for DNA sequencing

This article proposed the correctable decoding sequencing technology with conservative theoretical error rate of 0.0009%, and evaluated its robustness by simulation. This technology can provide a powerful new protocol for NGS platforms, enabling accurate identification of rare mutations in medicine.

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Thank you for this interesting contribution to Life Science Alliance. We are looking forward to receiving your revised manuscript. --High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title and running title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.
--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-sciencealliance.org/copyright-license-fee B. MANUSCRIPT ORGANIZATION AND FORMATTING: Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Reviewer #1 (Comments to the Authors (Required)): The Manuscript "Evaluation of the correctable decoding sequencing as a new powerful strategy for DNA sequencing" describes a theoretical approach to enhance accuracy of existing next generation sequencing platforms. The innovation it presents consist of replacing the single-base flows in conventional single nucleotide addition sequencing workflows with dual-base additions. This is not entirely new and will result in highly ambiguous sequencing results, that can only be interpreted by repetition of the sequencing workflow with different dual-base combinations. The approach presented here replaces the dual-base addition of two natural nucleotides with a mixture of a natural and a reversibly terminated nucleotide. Combined with regular deblocking flows to remove the terminators This will result in less ambiguous sequencing results that can be decoded by only two consecutive sequencing runs with the same template. The approach seems original and the claim for higher accuracy is justified. Some simulated results are included. I would support acceptance of the manuscript, after some minor revisions: (1) A software is mentioned that can simulate sequencing results as they would be expected from the described sequencing method. As the ability to correct sequencing errors is claimed, a reasonably large set of sequencing reads containing errors according to the typical error profile of one of the mentioned commercial systems should be generated and subjected to the proposed error correction. The outcome of this simulation should be shown to justify the claim of extraordinary sequence accuracies, eg by presenting a statitics about the raw error rate oft he simulatedv reads and a final error rate after algorithmic error correction. (2) The overall language quality of the manuscript is quite poor with a lot of grammar errors, incomplete sentences, missing words and incorrect expressions. Therefore language editing by a native or highly proficient english speaking person seems necessary.
Reviewer #2 (Comments to the Authors (Required)): The manuscript "Evaluation of the correctable decoding sequencing technology as a new powerful strategy for DNA sequencing" proposed the new protocol to reduce the error in existing NGS platforms. As there are so many applications of NGS, one of them is pharmacogenomics studies which are mainly based on SNPs. less error in sequencing is directly proportional to less false-positive SNPs and other variations, there is a need for such protocol which fulfill this requirement.

Major Issue
The overall manuscript is in good shape and provides a useful alternative, but, as the author proposed new software to analyze and assemble sequences how they claim that they reduce the shortcoming of NGS platforms. Here as per the manuscript, it seems that they relied on the same platform but proposed the new algorithm/ software to analyze sequence more accurately. They should clearly claim for a new protocol, not a platform.
There are a few minor queries as well -Author claims single-read information accuracy, what is the basis of this claim? -What will be standard sequence coverage for the proposed method? Reviewer #2: Comment 1: The overall manuscript is in good shape and provides a useful alternative, but, as the author proposed new software to analyze and assemble sequences how they claim that they reduce the shortcoming of NGS platforms. Here as per the manuscript, it seems that they relied on the same platform but proposed the new algorithm/ software to analyze sequence more accurately. They should clearly claim for a new protocol, not a platform.
Response: Thank you for the comments on the paper. Your advice is very good.
The approach we proposed is a new protocol, not a platform. We have changed in this manuscript.
Comment 2: Author claims single-read information accuracy, what is the basis of this claim?
Response: A template needs to be interrogated twice in the correctable decoding sequencing approach. Therefore, a completely aligned sequence of consecutive bases can be obtained only for two different sequential sequences with the same base deletion or insertion at the same base position (including homopolymer region), as well as with the same pattern and number of sequencing errors. In the simulation, we calculated a conservative theoretical error rate of 0.0009% (lower than that for Sanger sequencing). Thus, this proposed approach can be used to assess the correctness of a single sequencing read.
Comment 3: What will be standard sequence coverage for the proposed method?

Response: Unlike the current NGS, the template is interrogated via multiple parallel sequencing runs (not simple repetitions) with the correctable decoding
sequencing method, we can determine whether the original sequencing information is correct by aligning any two sets of sequencing information, and the corrected "alignment information" can be used as a read for sequence assembly. Therefore, theoretically, accurate sequence information can be obtained when the sequencing depth is 1×, thereby reducing the coverage required for assembly. In general, the sequencing depth refers to the coverage depth of the sequencing data on the genome. Coverage is the proportion of the genome that can be covered by sequencing data compared to the reference genome. For example, if the human genome is 3G, we measured and filtered 90G of clean data, then the sequencing depth is 90/3=30x, and the coverage is 80%.
Therefore, the specific coverage is related to the composition of the genome to be sequenced and fragmentation technology.
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Once again, thank you very much for your comments and suggestion.
Sincerely yours, Thank you for submitting your revised manuscript entitled "Evaluation of the correctable decoding sequencing as a new powerful strategy for DNA sequencing". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. The revised version of the manuscript "Evaluation of the correctable decoding sequencing as a new powerful strategy for DNA sequencing" describes a theoretical approach to enhance accuracy of existing next generation sequencing platforms. The innovation it presents consist of replacing the single-base flows in conventional single nucleotide addition sequencing workflows with dual-base additions. This is not entirely new and will result in highly ambiguous sequencing results, that can only be interpreted by repetition of the sequencing workflow with different dual-base combinations. The approach presented here replaces the dual-base addition of two natural nucleotides with a mixture of a natural and a reversibly terminated nucleotide. Combined with regular deblocking flows to remove the terminators. This will result in less ambiguous sequencing results that can be decoded by only two consecutive sequencing runs with the same template. The points raised during the first review have now been adequately treated and can in the present form be recommended for publication Reviewer #2 (Comments to the Authors (Required)): The authors include all the suggested points, and now the manuscripts seem in a good and acceptable format.