Evaluation of five commercial DNA extraction kits using Salmonella as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories
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Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost and has a faster turnaround time, in addition to the ability to assemble complete bacterial genomes. The quantity and quality of DNA required for Nanopore sequencing are greater than for Illumina, and the DNA extraction methods recommended for obtaining high-molecular-weight DNA are different from those typically used in diagnostic laboratories. Using a Salmonella isolate with a previously closed PacBio genome as a model Enterobacteriaceae organism, we evaluated the quantity, quality and fragmentation of five commercial DNA extraction kits. Nanopore sequencing performance was evaluated for the top three methods: Qiagen EZ1 DNA Tissue, Qiagen DNeasy Blood and Tissue, and a modified, in-house version of the MasterPure Complete DNA and RNA purification. To evaluate the effect of post-extraction DNA purification methods, we subjected extracted DNA from the three selected extraction methods to purification by AMPure beads or ethanol precipitation and compared these outputs with untreated DNA as a control. All methods are suitable for routine whole-genome sequencing (WGS), since all 60 replicates had very high genome recovery rates, with ≥98 % of the reference genome covered by mapped Nanopore reads. For 85 % of the replicates, assembly was able to produce a complete, circular chromosome using either Flye or Canu. In most cases, it is recommended to move directly from extraction to sequencing, as untreated DNA had the highest rates of genome closure regardless of extraction method. Using our evaluation criteria, the Qiagen DNeasy Blood and Tissue kit was found to be the best overall method due to its low cost, ability to scale from single tubes to 96-well plates, and high consistency in yield and sequencing performance.
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Thank you for addressing all comments raised by the reviewers and myself.
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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 made by the reviewers, I would like to suggest to include a diagram of the workflow with information what was evaluated at each step, this might help readers to follow the results better.
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Comments to Author
The manuscript presented evaluates various DNA extraction methods for use with Nanopore sequencing using the Rapid kit with a specific view as to how useful each is in the context of a diagnostic laboratory. A Salmonella enterica strain which has previously been sequenced to completion with high-quality combinatorial assembly using both Illumina and PacBio sequencing chemistry. Five different commercial extraction kits were tested here, spanning different approaches to DNA extraction, in addition, an altered method for one of these kits was trailed separately. The manuscript is generally well presented, and the results seem to be properly interpreted. All raw data in the form of sequencing reads are available online under bio-project: PRJNA768992. Each kit is evaluated in terms of the quality and …
Comments to Author
The manuscript presented evaluates various DNA extraction methods for use with Nanopore sequencing using the Rapid kit with a specific view as to how useful each is in the context of a diagnostic laboratory. A Salmonella enterica strain which has previously been sequenced to completion with high-quality combinatorial assembly using both Illumina and PacBio sequencing chemistry. Five different commercial extraction kits were tested here, spanning different approaches to DNA extraction, in addition, an altered method for one of these kits was trailed separately. The manuscript is generally well presented, and the results seem to be properly interpreted. All raw data in the form of sequencing reads are available online under bio-project: PRJNA768992. Each kit is evaluated in terms of the quality and quantity of the DNA extracted as well as the resulting sequence data obtained, from this it is concluded that the Qiagen DNeasy Blood and Tissue kit is the most suitable. Methodological rigour The methods for extraction of DNA as well as the evaluation of the different kits have been conducted toughly. The use of a scoring system to account for the various factors is a sensible method of comparison. The only methodological improvement I could suggest would be a statistical analysis of the data shown in figure 4 - I couldn't see a mention of this having been done but it would be relevant to indicate whether the differences in sequence output between extraction kits were significant. In addition, I would suggest that some sort of evaluation of the accuracy of the assemblies obtained would be relevant but not necessary to this work. If the analysis has been done already, the inclusion of information such as the number of SNPs detected in each assembly compared to the reference would be of interest since, as was mentioned, high coverage is often required to account for the higher error rate with Nanopore sequencing. 2. Presentation of results The results are generally well presented with DNA quantification, purity and fragment length all reported as well as the coverage, read length and N50 values for each extraction method. If possible, I believe two small changes could be made to the figures to improve readability. These are 1. Figure 1 included the average total DNA for each extraction method and 2. Table 3 showed "the percentage of replicates with complete chromosomes" instead of "number with incomplete chromosomes". There is potentially some confusion with the references to supplementary data in the manuscript. For example, data from supplementary file S2 is referred to as being both S1 and S2. Below I have outlined what I found to be the associations as best I could. Line 210: Data S1 - Manufacturers' protocols with modifications are described Is "Supplemental File 1" Lines 426, 429, 457: Data S1 - Assembly methods that generate circular chromosome Is "Supplementary File 2" Line 282: Data S2 - file containing accession numbers Is "Supplementary File 2" Lines 395, 397: Data S2 mean coverage grouped by extraction method I was unable to find the data this refers to Line 437: Data S2 mean read length and N50s Is "Supplementary File 2" Lines 443, 445 and 449: Data S2 coverage for data that produced incomplete chromosomes for Flye and Canu assemblies I was unable to find the data this refers to - figures are similar to what would be obtained by averaging the data given in "Supplementary File 2" but not quite the same as my calculations. Supplemental Figure 1 - I didn't find a reference to this figure, and it was lacking a legend. 3. How the style and organization of the paper communicates and represents key findings The key finding here is that the Qiagen DNeasy Blood and Tissue Kit without any post-extraction concentration is the best extraction method for Salmonella in a diagnostic laboratory setting. This is communicated clearly with caveats such as cost, hands-on time and batch size accounted for. 4. Literature analysis or discussion Due to the nature of this manuscript, the scope for analysis is limited, however, the relevant literature and explanation of the use and limitations of sequencing in a diagnostic setting are included. The results are appropriately evaluated and discussed in the context. 5. Any other relevant comments In general, I thought that this manuscript achieved the intended outcome of evaluating various extraction methods and that this will be relevant for diagnostic testing of Salmonella and probably more widely. This type of result is of value as it will allow those with fewer resources or time to make more informed decisions on methods. Though it may be possible for the findings here to be communicated in a slightly more concise way. For this type of article, a more succinct methods and results section, in particular, would increase readability. Typos - Line 40 "WGS" should be defined for the first use Line 158 carbapenam should be carbapenem Line 465 an incomplete assembly?
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
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Comments to Author
1. Line 29: change "times" to "time"? 2. Line 292 please elaborate what is stopOnLowCoverage 3. Line 311, if 77.8 ng/uL (as well as the following concentrations presented) is a mean of replicates, please present the standard deviation for each mean (or use any other values that can show the range or variation of the means) 4. Line 372, does this conclusion take the post-extraction concentration time into consideration? Since the yield of QIA is lower than required by ONT protocol, it for sure needs a post-extraction concentration step to increase the concentration of the DNA to ONT required threshold 54 ng/uL (400ng in total in 7uL). Please clarify it here for this statement. Although in the conclusion part the author claims that unpurified QIA extracts performed well in sequencing using lower amounts …
Comments to Author
1. Line 29: change "times" to "time"? 2. Line 292 please elaborate what is stopOnLowCoverage 3. Line 311, if 77.8 ng/uL (as well as the following concentrations presented) is a mean of replicates, please present the standard deviation for each mean (or use any other values that can show the range or variation of the means) 4. Line 372, does this conclusion take the post-extraction concentration time into consideration? Since the yield of QIA is lower than required by ONT protocol, it for sure needs a post-extraction concentration step to increase the concentration of the DNA to ONT required threshold 54 ng/uL (400ng in total in 7uL). Please clarify it here for this statement. Although in the conclusion part the author claims that unpurified QIA extracts performed well in sequencing using lower amounts of DNA than recommended by ONT, users may want to pursue the required quantity 400ng most of the time to optimize the sequencing results. 5. Line 389, the paper Subtyping Evaluation of Salmonella Enteritidis Using Single Nucleotide Polymorphism and Core Genome Multilocus Sequence Typing with Nanopore Reads | Applied and Environmental Microbiology (asm.org) (https://journals.asm.org/doi/10.1128/aem.00785-22) can be of help for the recommended sequencing depth of genome coverage for WGS with ONT data. 6. Line 392, the data yield or sequencing depth of each flow cell is dependent on several factors including input DNA quantity, length of each DNA read, number of DNA reads (can be calculated using input DNA quantity and N50 DNA length of all reads), initial number of active pores, please specify or discuss the potential influence of these factors to the final data yield along with the comparison between methods, otherwise, the comparison can be bias. 7. Please elaborate on this "Although ≥ 98% of the genome is represented by reads", what does "represented by reads" indicate?
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
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