Optimization of a DNA extraction protocol for improving bacterial and fungal classification based on Nanopore sequencing
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Ribosomal RNA gene amplicon sequencing is commonly used to evaluate microbiome profiles in health and disease and document the impact of interventional treatments. Nanopore sequencing is attractive since it can provide greater classification at the species level. However, optimized protocols to target marker genes for bacterial and fungal profiling are needed. To achieve an increased taxonomic resolution, we developed extraction and full-length amplicon PCR-based approaches using Nanopore sequencing. Three lysis conditions were applied to a mock microbial community, including known bacterial and fungal species: ZymoBIOMICS lysis buffer (ML) alone, incorporating bead-beating (MLB) or bead-beating plus MetaPolyzyme enzymatic treatment (MLBE). In profiling of bacteria in comparison to reference data, MLB had more statistically different bacterial phyla and genera than the other two conditions. In fungal profiling, MLB had a significant increase of Ascomycota and a decline of Basidiomycota, subsequently failing to detect Malassezia and Cryptococcus . Also, a principal coordinates analysis plot by the Bray–Curtis metric showed a significant difference among groups for bacterial ( P= 0.033) and fungal ( P= 0.012) profiles, highlighting the importance of understanding the biases present in pretreatment. Overall, microbial profiling and diversity analysis revealed that ML and MLBE are more similar than MLB for both bacteria and fungi; therefore, using this specific pipeline, bead-beating is not recommended for whole gene amplicon sequencing. However, ML alone was suggested as an optimal approach considering DNA yield, taxonomic classification, reagent cost and hands-on time. This could be an initial proof-of-concept study for simultaneous human bacterial and fungal microbiome studies.
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Thank you for addressing the concerns raised by the reviewers in your revised version. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.
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This is a study that would be of interest to the field and community. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments. In addition to the comments from the two reviewers, I would like to add the following In the introduction it appears very abrupt the way you switch from bacterial to fungal microbiome, I think it would be nicer to write a shared paragraph and then talk about the pecularites for each one. Somewhere you write fungal mycobiome, either remove fungal or write fungal microbiome, although a similar point was raised by the reviewers. I would also like to draw your attention to the comment of reviewer 2 regarding normalisation of the fungal data, please make sure you address this concern in your revised version.
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Comments to Author
In this manuscript, the authors explore the influence of three different lysis methods in the final DNA yield and bacterial and fungal classification in microbiome studies based on full-length amplicon Nanopore sequencing. Having said that, I do think that this is a protocol optimization. Therefore, I suggest rewriting the title. The research objectives are clearly stated, although to be completely met further replicates should have been included in the experimental design. Notwithstanding, the provided methods and results could be useful for the scientific community. Methods are adequately described to reproduce the experiments once suggestions below are implemented. One aspect that worries me is that the analysis of the mycobiome was not normalized (see my comments below). In general, the article is …
Comments to Author
In this manuscript, the authors explore the influence of three different lysis methods in the final DNA yield and bacterial and fungal classification in microbiome studies based on full-length amplicon Nanopore sequencing. Having said that, I do think that this is a protocol optimization. Therefore, I suggest rewriting the title. The research objectives are clearly stated, although to be completely met further replicates should have been included in the experimental design. Notwithstanding, the provided methods and results could be useful for the scientific community. Methods are adequately described to reproduce the experiments once suggestions below are implemented. One aspect that worries me is that the analysis of the mycobiome was not normalized (see my comments below). In general, the article is well written, and conclusions are sound by the presented results. Below, my suggestions for improvement of the manuscript are found: - Line 57: The microbiome includes the mycobiome, please rewrite and refer to bacterial and fungal microbiomes. - Line 242 and 279: Shannon Index does not measure the evenness, but the diversity within samples. - Lines 248-249: "With very low reads on MLB1_2, there was no subsampling for the 18S rRNA gene" Could the authors explain how do they normalize data instead? This is an important aspect to take into account. In this case there are different options: i) lower subsampling to the number of reads of the sample with the lowest number of reads; ii) remove that sample from the analysis; iii) normalize to sequencing depth (which could be the most recommendable). I think it is not acceptable that fungal microbiome is not normalized. Authors must at least comment this in the discussion. - Lines 295-297: I do not understand this. Due to intra-sample variation in DNA concentration, they decided to use just the initial sample. However the results always show two samples (which indeed are just a replicates from one sample) for each method. Could the authors explain this better? - Lines 307-310: Please, rewrite in past tense. - Figure 4B and 7B: "mean of beta-diversity distance" to what? I suppose this figure represents "mean of beta-diversity distance" to the reference. Please, write the correct label in the axis and figure caption. - Line 336: Try not to use the term "dramatically". - Line 428: I suppose the word "little" or "small" is missing between "very" and "proportion". -Lines 500-501: There's something strange in this sentence. It seems that there is a part missing. Take care of the punctuation and repeated "is". - Methods S1: I think that, to discard that the problem of not being able to sequence Malassezia is not the DNA extraction method per se, this extraction should have been made with the Kit used in the whole study (ZymoBIOMICS 96 MagBead DNA Kit) and not a different one.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
MANUSCRIPT ID ACMI-D-23-0217 - REVIEW REPORT TITLE Optimisation of a DNA extraction protocol for improving the bacterial and fungal classification based on Nanopore sequencing SYNOPSIS AND MAJOR COMMENTS The manuscript entitled "Optimisation of a DNA extraction protocol for improving the bacterial and fungal classification based on Nanopore sequencing" presents a well-structured overview of different methods for obtainment of high-quality metagenomic DNA for Nanopore-based microbiome surveys. The message is clear from the beginning of the manuscript and the methods are extremely well-detailed, which makes reading really enjoyable and engaging even for a non-specialist who might be interested in adapting their DNA extraction protocols. The hard work in getting all these protocols should be praised. …
Comments to Author
MANUSCRIPT ID ACMI-D-23-0217 - REVIEW REPORT TITLE Optimisation of a DNA extraction protocol for improving the bacterial and fungal classification based on Nanopore sequencing SYNOPSIS AND MAJOR COMMENTS The manuscript entitled "Optimisation of a DNA extraction protocol for improving the bacterial and fungal classification based on Nanopore sequencing" presents a well-structured overview of different methods for obtainment of high-quality metagenomic DNA for Nanopore-based microbiome surveys. The message is clear from the beginning of the manuscript and the methods are extremely well-detailed, which makes reading really enjoyable and engaging even for a non-specialist who might be interested in adapting their DNA extraction protocols. The hard work in getting all these protocols should be praised. This is evident not only by their description but also by the care taken by the authors to justify each one of the results with variations that could be explained by the different DNA extraction strategies investigated. All the results are comprehensible by both the main and supplementary figures. Thus, this current work has the potential to set up the scene for successful Nanopore sequencing of 16S rRNA and 18S rRNA gene regions in microbiome reports. The only section that could deserve more attention is Discussion. Authors tend to repeat Results and misplace them in this part of the manuscript as well. Here, it could be better to highlight the novelty and impact that the main conclusion of the work, the high efficiency of the ML method. DETAILED REVIEW Abstract Line 49 Replace "index" with "metric". Line 55 Add "taxonomic" before "classification". Line 57 Either remove "mycobiome" or stratify the microbiome components: "bacteriome", "archaeome" and "mycobiome". Line 59 To avoid repeating the same word present in the manuscript title, replace "Nanopore" with "third generation sequencing" or other equivalent term. Introduction Line 61 Missing the "Introduction" word headline for the section. Line 63 Replace "unculturable" with "not-yet culturable". Line 67 Complement the idea of this sentence, perhaps providing some examples or adding some broad statement, such as "for different pathological conditions". Also, write "modification" in the plural form: "modifications". Line 69 Amplicon sequencing (metataxonomics) is more cost-effective than shotgun metagenomics. The word "considerably" can be removed from this sentence. Line 73 Correct, it's "bacterial and archaeal" profiles. The 16S rRNA gene is useful for amplicon-based diversity surveys members of both domains, despite some discussion on the effectiveness to disclose the full community picture, depending on the targeted hypervariable region. Line 74 The authors might considering reviewing the statement on the taxonomic resolution of metataxonomics: it works just fine up to the genus level, not species of strain-specific level, that can be achieved by applying shotgun metagenomics. Line 81 Add some connective conjunction to initiate this paragraph, perhaps "In particular". Line 91 Correct: "are being used" for "have been used". Line 99 Capitalize "Nanopore". Line 120 Add an adjective or expression that helps the reader to understand that both the bacterial and fungal microbiota are being investigated. Something like "comprehensive" or "complete". Lines 128 and 134 Add a comma after "Irvine". Lines 129-130 Even though the references for those mock communities' 13templates are still based on the previous phyla name, the authors might consider updating them ("-ota" suffix). Line 132 Can the authors provide between parentheses the fungal species included in this mock community mix? Line 140 Specify what was used as the starting material instead of the microbial reference suspension for the negative control: ultrapure water? Line 154 Italicize "g". Lines 160-161 These sentences might be rephrased into a single one. Line 166 BioEdit reference? Line 168 Even though, the authors utilised the CLUSTALW web version, it's important to include the original reference. Line 169 Place "Figure S1" between parentheses. Lines 171-175 Summarize this information in a Supplementary Table and remove this stretch from the main text. Also, these were the ten fungal species that should have been mentioned before? Line 186 Capitalise the "Nanopore" word. Lines 187-188 Remove "forward primer" and "reverse primer". Line 189 It can be just mentioned "(Table S1)"; remove for oligonucleotide primer sequences" Line 190 this "in-house" protocol was developed in the current work or it was based on one previously described in the literature? If this is the case, refer to the preexisting work. Line 204 Remove the comma and just write "(Table S2)" at the end of this sentence. Line 207 Remove the comma and just write "(Table S1)" at the end of this sentence. Line 208 Add "gene" after "rRNA". Lines 221-222 Rewrite this sentence, in order to place between parentheses just the website, there is no need to cite here the date it was accessed, this can be left for the bibliography at the end of the manuscript. Line 226 The SRA link for the genome deposit can be removed from this sentence. Line 240 Include the reference for MicrobiomeAnalyst: Chong et al., 2020 (https://doi.org/10.1038/s41596-019-0264-1) Line 250 Remove the comma and just write "(Figure S4)" at the end of this sentence. Line 252 Rephrase the title of this section, perhaps "Targeted amplification for detection of Malassezia genus". Lines 253-254 Primers sequences can be removed from the main text and included in Table S1, such as done for the other oligonucleotide sequences. Line 261 Remove the comma and just write "(Figure S2)" at the end of this sentence. Additionally, specify if the negative control included was the same as described in the previous section ("Amplification for Nanopore sequencing"). Lines 271-271 Remove the comma and just write "(Figure S3)" and "(Figure S4)" in this sentence. Line 279 Correct: "Chao1". Line 284 Include MicrobiomeAnalyst reference. RESULTS Line 294 Write "p" in italics. Line 304 Write "rRNA gene" after "16S" and "18S". Line 328 Remove the comma and just write "(Figure S7)" at the end of this sentence. Figures 2 and 3 Write "sp." in the regular form, not in italics. Figures 4 and 7 Write "p" in italics (below "PERMANOVA"). Also, provide the stress value for these ordination plots. Line 361 Remove the comma and just write "(Figure S1)" at the end of this sentence. Line 366 Write "(Figure S2A") after the primer set between parentheses. Line 367 Remove the extra dot. DISCUSSION Line 389 Add "rRNA gene" after "16S". Line 411 Capitalize "Illumina". Line 426 Write in the following manner: "(Figure S2A)". Lines 431-432 Remove the comma and just write "(Figure S2B and S3)" at the end of this sentence. Line 460 Write "p" in italics. Lines 475-483 This paragraph should be placed in the Results section, nothing really discussed here. Lines 489-503 Split into two sentences, there is a misplaced "." in line 500, maybe restructure this stretch around this sentence.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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