High-Throughput Isolation of Nucleic Acids from Soil

Round 1

Reviewer 1 Report

This manu reports a study to develop a high-throughput method to extract soil DNA, based on the BioSprint 96 platform and compared it with two 16 commercial column-based kits and another automated procedure. The results seem promising.

I am not an expert in soil DNA extraction, though we work on soil DNA everyday. I am not able to evaluate the novelty of the method. But to me the current method is cheap and efficient. A high-throughput method may be useful in some commercial companies and large scale DNA studies.

Basically the experimental part is well designed, they determined the quality, quantity and specific genes.

A major concern is the language quality. I would suggest the authors polish the language carefully before sending it for review.

Line 13, this sentence is not readable. A key step is... challenging...being.., what does that mean?

Line 23, --->numbers of samples

Line 28, --->soil is....diversity. Its...

Line 35, update the number of 99%. Currently this have been improved drastically

Lines 91,96, 110, normally we do not need to mention a table or figure deliberately. This is a waste of words. Describe the results directly.

Line 129, protocol1,--,check the space

Author Response

December 16, 2019

Dear Reviewer 1,

I hereby provide the responses to your comments to the manuscript entitled "High-throughput isolation of nucleic acids from soil" (Manuscript ID: 654612) .

Thank you for the thoughtful review of the manuscript. All comments and suggestions have been considered and written in red bold on the manuscript. Modified words, punctuation, minor revisions etc. were accepted and inserted in the text. To avoid word repetitions and/or for clarity, some modification was necessary before and after the revised or deleted sentences.  Authors' Response are listed in the attached file.

Thanks again for your help and encouragement.

Best Regards

Piergiorgio Stevanato

Author Response File: Author Response.pdf

Reviewer 2 Report

In the manuscript High-throughput isolation of nucleic acids from soil by Claudia Chiodi and collaborators, the authors compare different DNA extraction methods to find out the most effective one considering yield and purity of DNA and price and time per sample. The topic of the submitted manuscript is highly relevant as DNA extraction is one of the most important steps in the culture-independent methods. The manuscript was quite short and concise, but there were quite many parts which need further explanation or even additional analyses.

In the introduction, you should include many newer references. First seven references were more than 12 years old and in such a fast developing field as an environmental microbiology field is, you need to consider really last results from this field.

Lines 82-84 and lines 87-89: You state that there were no differences between yield of BioSprint, QIASymphony and FastDNA and Powersoil extraction and purification protocols (P>0.01). The same is repeated few lines later (No statistically significant differences were highlighted in the level of purity of DNA extracted with the four protocols (P>0.01)). My question comes from the Table 1 as I can see there the differences indicated with different letters. How do you comment that?

Lines 84-85 and lines 90-91: Between which soils and using which method no statistically significant differences were observed? Between all? Seems unlikely as the yield or purity of some are quite different while comparing three soils.

Line 91: “S1 presented the highest yield and purity.” – Please specify the best purity range also regarding A260/280.

As I started reading from the beginning of manuscript and the Material and methods section is in the end, then some parts have to be rewritten that way the manuscript is complete and understandable. In the results section, you need to specify at some point the soil types and show the physico-chemical properties of the soils or at least indicate where you can see some information more about these. This is important, because for example for me very many critical questions arisen, but later I got some answers to them from the methods section at least to some extent.

You write about Ct values and you compare them in section 2.2. Determination of DNA quantity by qPCR, but I could not see any Ct values in the manuscript. Please change these thing accordingly so that there are Ct values shown or compare gene copy numbers.

Quite many confusing sentences in the Discussion (lines 128-132, 136-138, etc.). Please rephrase them in easier way.

That is not a correct way to say it (lines 168-170). I can see statistically significant differences between all of these parameters (yield, purity and amplifiability, see Tables 1 and 2).

Final soil samples were dried at room temperature for 48 hours, crushed and sieved (Ø 0.5 mm). Samples were stored at -80 °C before proceeding with DNA extraction.” - It is completely insane to dry the samples before DNA extraction, when we want to say later something meaningful for the microbial community in these soils. The soil community and abundances of different genes may have been totally changed and there is no biological meaning in the end. Moreover, why is that sieving necessary?

Lines 257-259. We cannot say that only lower Ct values are showing the quality of qPCR. There may be false amplification of wrong product, primers may produce some false products, efficiency of amplification, etc.

Amplicons length of qPCR primers used was quite long. If the amplicon length is shorter, are the results of this manuscript still relevant?

Why to use functional primers as it is widely known that functional primers usually have very many drawbacks in both main criterias (universality and specificity)? Why not to use primers with less problems (e.g., primers of bacterial and archaeal specific 16S rRNA genes)?

Where from these equations are coming to calculate gene copy numbers? Seems like efficiency of amplification has not taken into account. It is crucial to take efficiency of amplification into account, especially in case of functional genes. For calculation, you can use widely accepted protocol - see supplementary materials and section of Quantitative PCR in this publication: https://www.nature.com/articles/s41598-018-23032-y#additional-information.

"Values considered for every determination were: mean, standard deviation and standard error of the mean." - What that sentence mean?

Conclusions. “… while keeping performances comparable to those of common manual kits” - For me, it feels very arbitrary conclusion as from your results it seems that I will lose to some extent in yield, purity and amplifiability, although I agree that I will win in time and cost. What is the optimum solution between these parameters - this is the question?

Question about title of manuscript also. Is present title suitable as the experiment/test is quite small and you use only three different soils, but title is broader in every way?

Other comments:

There were quite many spelling and grammar mistakes throughout the manuscript (very many typos even in the abstract! – see lines 20, 23, etc.).

Some abbreviations were not explained (e.g. see line 147).

Tables 1, 2 and 4 are missing n-values. Specify that standard errors are shown.

Merge two tables into one big table under Table 2. Table 2 is missing standard errors.

What program was used for which gene? (line 254)

Author Response

Dear Reviewer 2, 

I hereby provide the responses to your comments to the manuscript entitled "High-throughput isolation of nucleic acids from soil" (Manuscript ID: 654612) .

Thank you for the thoughtful review of the manuscript. All comments and suggestions have been considered and written in blue bold on the manuscript. Modified words, punctuation, minor revisions etc. were accepted and inserted in the text. To avoid word repetitions and/or for clarity, some modification was necessary before and after the revised or deleted sentences.  Authors' Response are listed in the attached file.

Thanks again for your help and encouragement.

Best Regards

Piergiorgio Stevanato

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript by Chiodi et al. shows the results of 5 independent DNA isolation methods. While there are no major differences between DNA isolation kits (besides crude DNA method) there is a difference in time and cost. Overall, this paper does not discover any novelty, as it is rather expected than automated, robotic system (as this Biosprint) will have lower cost and time per sample. This is a definition of automation. Anyway, this paper may be useful for someone who seeks how to efficiently and economically isolate thousands of soil DNA samples and hence I can endorse it for publication once the authors can clarify their qPCR results. Some minor comments below.

Major:

qPCR – was the DNA amount for each sample standardised or simply 1 ul of each sample was added? If the DNA amount (mass rather than volume) was different between samples then this experiment is of low value. I assume you want to present the amplification ability of each DNA isolation method. Are you suggesting than “crude DNA” with ~50 fold higher concentration of DNA (or at least the total isolated amount – here I assume that all methods resulted in the same total volume) showed 5 order of magnitude lower qPCR efficiency for amoA gene? What were the Ct values for these samples? If the Ct values reached 25 or more cycles then you just don’t have any amplificable materia and/or there are inhibitors of the amplification reaction. I doubt that you can calculate both 10^1 and 10^7 values from the same qPCR calibration curve within a reasonable number of amplification cycles.

The other problem with your qPCR experiment is the difference between amoA and nosZ amplification efficiency. Why Crude DNA seem not to have any amoA while has reasonable amounts of nosZ? To me it seems that your amoA qPCR for crude DNA just failed.

Moreover, please add some units to your values. Is your output presented per 1ul of DNA, per 1 gram of soil? Do you have some SE values or was it a single qPCR run per sample or group of samples.

minor:

line 91: either sample S1 has the highest yield and purity (statistically) or not as the line 90 states. Please remove line 91 or justify.

Table 1 – please state what do the error mean – SD or SE?

line 168-169. I am a bit confused here. If you say that all the tested method (aside the crude DNA method) gave you similar results (yield, purity and amplification) while the only difference is in time and cost, surely you can get that information from kits protocols. I am not criticizing the point of doing all this work, however, I would underline that you have shown a great similarity between the kits and the only difference is that some of them can be automated with this Biosprint or QIASymphony machine (seems that Qiagen charges a lot for their kits, though).

Author Response

Dear Reviewer 3, 

I hereby provide the responses to your comments to the manuscript entitled "High-throughput isolation of nucleic acids from soil" (Manuscript ID: 654612) .

Thank you for the thoughtful review of the manuscript. All comments and suggestions have been considered and written in green bold on the manuscript. Modified words, punctuation, minor revisions etc. were accepted and inserted in the text. To avoid word repetitions and/or for clarity, some modification was necessary before and after the revised or deleted sentences.  Authors' Response are listed in the attached file.

Thanks again for your help and encouragement.

Best Regards

Piergiorgio Stevanato

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Language quality is improved largely for this version. Yet spell check is still required before possible publication.

e.g., line 75, are reported---> is reported; line 76 materials and methods---->Materials and

Author Response

Kind reviewer 1,

thank you again for your suggestions. Mistakes have been corrected (bold red) and more imporvements to the language have been brought.

Reviewer 2 Report

I really appreciate that you have considered all the comments and I feel satisfied that many things have been changed into more correct way.

“Contrary to this reviewer’s assumption, drying soils is the standard procedure for DNA based analyses” - I agree that probably in light of this research, it does not matter a lot if you have dried your samples before DNA extraction or not. But when we consider biological aspect, then drying will affect further results (Castaño, C., Parladé, J., Pera, J., De Aragón, J.M., Alday, J.G. and Bonet, J.A., 2016. Soil drying procedure affects the DNA quantification of Lactarius vinosus but does not change the fungal community composition. Mycorrhiza, 26(8), pp.799-808.). Actually, it seems that pre-treatment of DNA extraction depends on protocols and methods you use (you can use wet samples also). See also this review: Lear, G., Dickie, I., Banks, J., Boyer, S., Buckley, H.L., Buckley, T.R., Cruickshank, R., Dopheide, A., Handley, K.M., Hermans, S. and Kamke, J., 2018. Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. New Zealand Journal of Ecology, 42(1), pp.10-50A.

“This argument does apply to each and every quantitative PCR experiment in the world. While those issues are a general occurrence and are always in the background of any amplification experiment, the Ct value is nevertheless the plain output of qPCR on which its whole principle is based. In other words, we agree but we intended those eventualities as implicit.” – I wanted to point out just some drawbacks, but what I meant here more is that please rephrase it because otherwise it seems that only Ct value matters. If we do analysis with real samples, then we consider much more parameters (melt curves etc.) to evaluate the success of our amplification. This is why we cannot say that only lower Ct values are showing the quality of qPCR. For example, we can also get very low Ct values, when we use primers which amplify wrong product etc.

"Actually we do not understand the concern or the question" - Here I meant that the amplicon length can change amplifying etc, but most of these things are more question of suitable qPCR program.

Caption of Table 1 can be made more readable. And Table 1 can be made also with less repetitions.

"Merge two tables into one big table under Table 2." - Actually, I meant that here initially:

Author Response

Kind reviewer 2,

Thank you again for your precious comments. We agree with you and we will take your suggestions into consideration for further works. We modified the manuscript accordingly. 

Particularly, we added a sentence (line 257) to clarify your doubts regarding the Ct. We also modified the table as you suggested, our apologies for the misunderstanding.

Best regards