Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing
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Abstract
Background
Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types.
Results
We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20–25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20–25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield.
Conclusion
We provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.
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Studies
This work has been published in GigaScience Journal under a CC-BY 4.0 license (https://doi.org/10.1093/gigascience/giac068) and has published the reviews under the same license.
**Reviewer 1 Tomas Sigvard Klingström, **
As a researcher who may occasionally use long read sequencing technique for projects it is immensely helpful to get an insight into the experience accumulated through work related to the Vertebrate Genomes Project (VGP). My personal research interest on the subject is more on understanding why and how DNA fragment during DNA extraction. Due to my work in that area I have one key question regarding the interpretation of the data presented in figure 2 and then a number of suggestions for minor edits. The answer on how to interpret figure 2 may require some minor edits but the article is regardless of this a …
Studies
This work has been published in GigaScience Journal under a CC-BY 4.0 license (https://doi.org/10.1093/gigascience/giac068) and has published the reviews under the same license.
**Reviewer 1 Tomas Sigvard Klingström, **
As a researcher who may occasionally use long read sequencing technique for projects it is immensely helpful to get an insight into the experience accumulated through work related to the Vertebrate Genomes Project (VGP). My personal research interest on the subject is more on understanding why and how DNA fragment during DNA extraction. Due to my work in that area I have one key question regarding the interpretation of the data presented in figure 2 and then a number of suggestions for minor edits. The answer on how to interpret figure 2 may require some minor edits but the article is regardless of this a welcome addition to what we know about good practices for DNA extraction generating ultra high molecular weight DNA. It should also be noted that the DOI link to Data Dryad seems broken and I have therefore not look at the supplementary material. In figure 2 the size distribution of DNA fragments is visualized from the different experiments. Most of the fragment distributions look like I would have expected them based on the work we did in the article cited as nr 25 in the reference list. However the muscle tissue from rats and the blood samples from the mouse and the frog indicates that there may be a misinterpretation in the article regarding the actual size distribution of fragments which needs to be looked in to. Starting with the mouse plots and especially the muscle one. There must either have been a physical shearing event that drastically reduced the size of DNA (using the terminology from ref 25 this would mean that physical shearing generated a characteristic fragment length of approximately 300-400 kb), or the lack of a sharp slope on the rightmost side of the ridgeline plot is due to the way the image was processed. All other animals got a peak on the rightmost side of the ridgeline plot and the agarose plug should, based on the referenced methods paper [7], generate megabase sized fragments which far exceed the size of the scale used in figure 2. I would presume these larger fragments would get stuck in or near the well which makes it easy to accidentally cut them out when doing the image analysis step which may explain their absence in the mouse samples. This leads me to the conclusion that the article is well designed to capture the impact of chemical shearing caused by different preservation methods but would benefit from evaluating whatever figure 2 properly covers the actual size distribution of fragments or only covers the portion of DNA fragments small enough to actually form bands on the PFGE gel with a substantial part of the DNA stuck in or near the well. The frog plot is a good example of how this may influence our interpretation of the ridgeline plots. If the extraction method generate high-quality DNA concentrated in the 300-400 kb range then there must be something very special with the frog DNA from blood as there is a continuous increase in the brightness all the way to the edge of the image. This implies that the sample contains a high amount of much larger DNA fragments than the other samples. I find this rather unlikely and if I saw this in my own data I would assume that we had a lot of very large DNA fragments that are out of scale for the gel electrophoresis but that in the case for the frog blood samples many of these fragments have been chemically sheared creating the "smeared" pattern we see in figure 2.
Minor edits and comments: Dryad DOI doesn't work for me. Figure 1 - The meaning of x3 and x2 for the turtle should be described in the caption. Figure 2 - Having the scale indicator (48.5. 145.5 etc) at the top as well as the bottom of each column would make it quicker to estimate the distribution of samples. The article completely omits Nanopore sequencing, is there a specific reason for why lessons here are not applicable to ONT? There is a very interesting paragraph starting with "The ambient temperature of the intended collecting locality should be a major consideration in planning field collections for high-quality samples. Here we test a limited number of samples at 37°C to". Even if the results were very poor information about the failed conditions would be appreciated. What tissues/animals did you use, did you do any preservation at all for the samples and did you measure the fragment length distribution anyway? Simply put, even if the DNA was useless for long read sequencing it is an interesting data point for the dynamics of DNA degradation and a valuable lesson for planning sampling in warm climates.
Re-review:
All questions and commends made in my first review are now resolved. I understand the thought process behind the first cropping of figure 2 but appreciate the 2nd version as it makes it easier for researchers with a limited understanding of the experiment to interpret the data.
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Abstract
**Reviewer 2. Elena Hilario **
I am glad to have been selected as reviewer for the manuscript "Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing" by Dahn and colleagues. The manuscript reports a detailed guide on the effect of preservation methods on the quality of the DNA extracted from a wide range of animal tissues. Although the work is only focused on vertebrates, it is a great foundation to conduct similar studies on plants, invertebrates and fungi, for example. Although the effectiveness of the tissue/preservative combination was only tested with the preparation of long range libraries, it would have been useful to select one or two cases for long range sequencing (PacBio or Oxford Nanopore) to explore the impact of the different QC parameters measured in this …
Abstract
**Reviewer 2. Elena Hilario **
I am glad to have been selected as reviewer for the manuscript "Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing" by Dahn and colleagues. The manuscript reports a detailed guide on the effect of preservation methods on the quality of the DNA extracted from a wide range of animal tissues. Although the work is only focused on vertebrates, it is a great foundation to conduct similar studies on plants, invertebrates and fungi, for example. Although the effectiveness of the tissue/preservative combination was only tested with the preparation of long range libraries, it would have been useful to select one or two cases for long range sequencing (PacBio or Oxford Nanopore) to explore the impact of the different QC parameters measured in this study.
Minor comments and corrections are included in the file uploaded
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