Comparative evaluation of soil DNA extraction kits for long read metagenomic sequencing

  1. Harry T. Child
  2. Lucy Wierzbicki
  3. Gabrielle R. Joslin
  4. Richard K. Tennant

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Abstract

Metagenomics has been transformative in our understanding of the diversity and function of soil microbial communities. Applying long read sequencing to whole genome shotgun metagenomics has the potential to revolutionise soil microbial ecology through improved taxonomic classification, functional characterisation and metagenome assembly. However, optimisation of robust methods for long read metagenomics of environmental samples remains undeveloped. In this study, Oxford Nanopore sequencing using samples from five commercially available soil DNA extraction kits was compared across four soil types, in order to optimise read length and reproducibility for comparative long read soil metagenomics. Average extracted DNA lengths varied considerably between kits, but longer DNA fragments did not translate consistently into read lengths. Highly variable decreases in the length of resulting reads from some kits were associated with poor classification rate and low reproducibility in microbial communities identified between technical repeats. Replicate samples from other kits showed more consistent conversion of extracted DNA fragment size into read length and resulted in more congruous microbial community representation. Furthermore, extraction kits showed significant differences in the community representation and structure they identified across all soil types. Overall, the QIAGEN DNeasy PowerSoil Pro Kit displayed the best suitability for reproducible long-read WGS metagenomic sequencing, although further optimisation of DNA purification and library preparation may enable translation of higher molecular weight DNA from other kits into longer read lengths. These findings provide a novel insight into the importance of optimising DNA extraction for achieving replicable results from long read metagenomic sequencing of environmental samples.

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  2. Access Microbiology

    Comments to Author

    Dear authors, The response to the comments are satisfactory, hence I would like to recommend the manuscript to proceed for publications.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    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

  3. Access Microbiology

    Comments to Author

    This study discusses a significant concern in soil microbiology regarding long-read metagenomic sequencing. It compares five common DNA extraction kits and four soil types, assessing which kit could give better NGS results. The authors addressed all three reviewer comments, thus improving the manuscript further. Overall, the study now contains all methods required for reproducibility (previously a major concern of the reviewers), and the authors have further developed their discussion and conclusions sections. Please see my minor comment below: Line 128 - "roots were removed from soil samples before being stored at 4 ˚C": Could you include information on how the roots were removed (sieved?) and how long the soil samples remained at 4oC before further analysis?

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    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

  4. Access Microbiology

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    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

  5. Version published to 10.1099/acmi.0.000868.v3 on Access Microbiology
  6. Version published to 10.1099/acmi.0.000868.v2 on Access Microbiology
  8. Access Microbiology

    Comments to Author

    Dear author, I have gone through your manuscript and I feel there are few major correction that has to be modified. The methods section does not provide sufficient detail on the specific protocols used for each DNA extraction kit. This includes information on the exact steps, incubation times, and any modifications made to standard protocols, which are crucial for reproducibility (Page 8). There are insufficient information regarding the sample size and the number of replicates for each extraction method. Clear reporting of these details is essential to assess the statistical power and reliability of the results (Page 34). There is no adequate discription regarding the environmental conditions under which soil samples were collected, such as temperature, moisture, and time of collection. These factors can significantly influence microbial community composition and DNA quality (Page 34). The methods section does not clearly outline the statistical methods used for data analysis, including how beta diversity was calculated and the specific tests employed for comparing microbial communities. Detailed statistical methods are necessary for validating the findings (Page 25). The results does not discuss the limitations or potential biases associated with each extraction kit, which could affect the interpretation of the results. Acknowledging these limitations is important for contextualizing the findings (Page 35).

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    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

  9. Access Microbiology

    Comments to Author

    1. The number of study samples is too low. It's recommended to use more diversified soil samples. 2. This study showed lacking about the suitability of others type of soil samples. 3. There is no interpretations about the soil bacterial composition.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    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

  10. Access Microbiology

    Comments to Author

    This interesting study tests five common DNA extraction kits against four different soil types (two natural and two agricultural) to evaluate which kit could be best for long-read metagenomic sequencing. These findings prove that standardisation and optimisation during DNA extraction are required to better understand the soil microbiome. The manuscript is well-written and has interesting results that I will definitely consider during my next round of DNA extractions. Please see my comments below: Line 61 - "the remote location of sampling sites": I'm unsure how sampling site remoteness contributes to methodological difficulties and metagenomics. Do the authors mean in terms of sample collection? If yes, wouldn't this be true no matter where the sampling site is? Line 136 - "A horizon": Could you explain this? I see it in Line 205 of the results, "Topsoil (A horizon)", but the Methods section appears first, so it should be explained here instead. Lines 137-138: Could you expand on how soil pH, field capacity, organic matter and carbon content were measured? More details are needed for reproducibility. Line 145 - Table 1: SD values are mentioned below the pH. Is this standard deviation? Of how many measurements? Line 145 - Table 1: Woodland NATMAP soil association is "Deep loam OR Stony sandy over sandstone?". Is the question mark needed? Line 151 - "Aforementioned order": Where is this mentioned? The names of the extraction kits should be mentioned within the text, not only in Table 2. Line 166 and throughout the results: Was the A260/A230 ratio measured? How does this compare between each kit? The PowerSoil kits are known for their low A260/A230 ratio, so it would be interesting to see how the other kits compare. Also, I wonder if this ratio would correlate with the decrease in DNA length in natural soils (heath and woodland; Figure 4f) since they both contain higher organic matter than the agricultural soils (Table 1). Results: Could you add a comment about your negative controls? Figure 6 and other NMDS plots: What stress values do these plots have? Could they be added to the figures? Venn diagrams: These figures are very visually appealing but make the data hard to understand. Could a different visualisation method be used instead? A dot plot, maybe? Lines 517-522: Do the authors have any suggestions on how to recover more of the fungal community? Line 567, "lowest PCR efficiencies": I'm sorry if I missed it, but where are PCR efficiencies in the results? Or is this referring to the short sequences primarily consisting of PCR artefacts (Line 307)?

    Please rate the manuscript for methodological rigour

    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

  11. Version published to 10.1099/acmi.0.000868.v1 on Access Microbiology