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. We find that no single method is best for all cases. Instead, the optimal storage and extraction methods vary by taxa, by tissue, and by down-stream application. Therefore, 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 generate the uHMW DNA needed for the high-quality reference genomes for Phase 1 of the Vertebrate Genomes Project (VGP), whose ultimate mission is to generate chromosome-level reference genome assemblies of all ~70,000 extant vertebrate species.

Sample collection. We collected samples from species representing major taxonomic classes of vertebrates, i.e. house mouse (Mus musculus), zebra finch (Taeniopygia guttata), Kemp’s Ridley sea turtle (Lepidochelys kempii), painted turtle (Chrysemys picta), American bullfrog (Rana catesbeiana), and zebrafish (Danio rerio). All animal handling and euthanasia protocols were approved by the Institutional Animal Care and Use Committees or equivalent regulatory bodies at the respective facilities: The Rockefeller University for the frog and bird samples; the Max Planck Institute for the mouse samples; the University of Toronto for the painted turtle sample; the Wellcome Sanger Institute for the fish samples; and the New England Aquarium rehabilitation facility for the sea turtle samples.

For this experiment, tissue samples were collected as available at facilities already handling the target species. The tissue types collected per species are as follows: mouse, spleen and muscle; zebra finch, whole blood and muscle; sea turtle, isolated red blood cells (RBCs); painted turtle, whole blood and muscle; bullfrog, whole blood and muscle; zebrafish, whole body, ovary, and muscle. For all species except the sea turtle and the fish, samples originate from a single individual. In the sea turtle set, duplicate samples were obtained from three individuals. In the fish set tissue samples in some cases originated from different individuals, as their small body size does not allow for sufficient amounts of tissue from a single specimen.

Each taxon required a slightly different handling procedure. All samples except for those from sea turtles were sourced from captive individuals humanely euthanized in a laboratory setting with approved protocols cited below. All soft or fibrous tissue samples were collected in small 20–30 mg pieces until each 2 mL tube had roughly 50–100 mg total to allow for full penetration of the preservative. Mice were euthanized by CO₂ treatment in a GasDocUnit (Medres Medical Research GmbH, Cologne, Germany) following the instructions of the manufacturer (DD24.1-5131/451/8, Landesdirektion Sachsen). Skeletal muscle and spleen samples were then dissected and placed in standard cryotubes. Birds were euthanized via isoflurane overdose, and whole blood was collected into chilled sodium heparin-treated 1.5 ml microfuge tubes (IACUC #19101-H). Then 25–50 µL was immediately aliquoted into cryotubes. Sea turtle RBC samples were collected from wild individuals undergoing medical treatment by drawing whole blood into 2 mL sodium heparin-treated collection tubes and then spinning down to separate RBCs from plasma. RBCs were then aliquoted into sodium heparin-treated tubes. Painted turtle samples were collected from one individual euthanized via decapitation as part of another study (AUP 20012070). Painted turtle muscle samples were immediately taken from the pectoral girdle and whole blood was drawn from the heart before placement in standard cryotubes. Frog samples were sourced from one captive adult purchased from Rana Ranch in Twin Falls, Idaho, USA. The frog was euthanized using an intracoelomic injection with Euthasol™ or Fatal-Plus™ (pentobarbital and phenytoin) at a dosage of 100 mg/kg. After confirming that a deep plane of anesthesia was reached, the frog was rapidly and doubly pithed cranially and spinally, then decapitated (19085-USDA). Frog muscle tissue samples were immediately taken from the rear legs and blood was drawn from internal veins before placement in standard cryotubes. We extracted fish samples from multiple lab-raised individuals. To euthanize the fish, we used tricaine and then the brain was destroyed with a scalpel (PPL No.70/7606). We collected white muscle and ovary samples which were dissected out and placed into 2 ml cryotubes immediately after euthanasia. Fish whole-body samples were taken by removing the head, intestines, and swim bladder of individual fish and placing the remaining tissue into a cryotube. 

Preservation treatments. A total of 140 freshly collected samples were subjected to different preservation and temperature treatments to test common preservation methods under simulated field or lab conditions, with flash-frozen samples being used as baseline controls. Preservation method treatments refer to the preservative agent applied directly to the sample before ultra-cold (–80°C) storage; temperature treatments refer to the temperature exposed and the amount of time the sample remained at that temperature before ultra-cold storage. 

All temperature treatments were applied immediately upon dissection of the material and placement into specimen tubes. Samples were exposed to temperature treatments of varying lengths of time in refrigeration (4°C), room temperature (20–25°C), and elevated temperature simulating a hot climate in an incubator to simulate field conditions in a tropical climate (~37°C). All temperature conditions tested and the samples to which they were applied are as follows: control condition submerged in liquid nitrogen from dissection to ultra-cold storage (all tissue types and species), 6 hr at 4°C (frog blood and muscle, bird blood and muscle, painted turtle blood and muscle, sea turtle RBCs), 16 hr at 4°C (mouse spleen, fish whole body), 1 day at 4°C (fish ovary), 1 week at 4°C (mouse muscle, frog blood and muscle, bird blood and muscle, painted turtle blood and muscle), 1 day at room temperature (fish muscle and ovary), 1 week at room temperature (mouse muscle, frog blood and muscle, bird blood and muscle, painted turtle blood and muscle, sea turtle RBCs, fish muscle and ovary), 4 weeks at room temperature (fish muscle and ovary), 5 months at room temperature (sea turtle RBCs), and 1 week at  37°C (mouse muscle). Storage time at –80°C after treatment and before DNA extraction varied slightly between samples, but such variation is expected to have a negligible impact on sample quality.

The preservation methods tested here include flash-freezing in liquid nitrogen, no added preservative agent, 95% EtOH, 20–25% DMSO-EDTA (DMSO), DNAgard, Allprotect, and RNAlater. Our DMSO recipe was 20–25% DMSO, 25% 0.5 M EDTA, remaining 50–55% H2O, saturated with NaCl. Flash-freezing, EtOH, and DNAgard were tested on all included species and tissue types. DMSO was tested on all species and tissue types except sea turtle RBCs. No preservative treatments were tested on bullfrog blood, bird blood, painted turtle blood, and sea turtle RBCs. Allprotect was tested on mouse spleen and muscle and fish body. RNAlater was tested on fish ovary and muscle samples. 

To gain insights into variation within these treatments, isolated RBC samples were collected from three different sea turtle individuals and processed separately as biological and technical replicates. The third replicate had insufficient material to test all treatments.

DNA extraction. We extracted DNA from all tissue samples using the agarose plug protocol as below, at VGP data production hubs at the Rockefeller University, Wellcome Sanger Institute, and MPGI Max Planck Institute Dresden. This method was established, at the time of this experiment, as standard protocol for long-read sequencing in all VGP projects. From each tissue sample, a 30–40 mg piece was weighed and then processed using the Bionano Prep™ Animal Tissue DNA Isolation Fibrous Tissue Protocol (Bionano document number 30071) and Soft Tissue Protocol (Bionano document number 30077). Briefly, the fibrous tissue (muscle, whole) pieces were further cut into 3 mm pieces and fixed with 2% formaldehyde and Bionano Prep Animal Tissue Homogenization Buffer. Tissue was blended into a homogenate with a Qiagen Rotor-Stator homogenizer and embedded in 2% agarose plugs cooled to 43°C. Plugs were treated with Proteinase K and RNase A, and washed with 1X Bionano Prep Wash Buffer and 1X TE Buffer (pH 8.0). DNA was recovered with 2 μl of 0.5 U/μl Agarase enzyme per plug for 45 minutes at 43°C and further purified by drop dialysis with 1X TE Buffer. The soft tissue (spleen, ovary) pieces were further cut into 3 mm pieces and then homogenized with a tissue grinder followed by a DNA stabilization step with ethanol. The homogenate pellet was then embedded in 2% agarose plugs as in the fibrous tissue protocol above. For blood samples, DNA was extracted from whole blood or RBCs following the unpublished Bionano Frozen Whole Nucleated Blood Stored in Ethanol – DNA Isolation Guidelines. The ethanol supernatant was removed, and the blood pellet was resuspended in Bionano Cell Buffer in a 1:2 dilution. For samples that freeze solidly at –80°C, tubes were thawed at 37°C for 2–4 minutes. The same Bionano guidelines for nucleated blood in ethanol were modified by adding 1–2 additional centrifugation steps at 5,000X g for 10 min prior to removing DNAgard supernatant and homogenizing blood cells in Bionano Cell Buffer in a 1:2 dilution. All samples were mixed with 36 µl agarose and placed in plug molds following the animal tissue protocol.

Assessing sample fragment size distributions. Fragment length distributions of samples were measured with at least one of two available methods: Pulsed-field Gel Electrophoresis (PFGE, n = 102) or the Agilent Femto Pulse system (FEMTO, n = 108). PFGE was performed using the Sage Science™ Pippin Pulse gel system with the Lambda PFG Ladder (New England Biolabs). Analysis of FEMTO outputs was carried out in the ProSize Data Analysis Software.

Hi-C library preparation and sequencing. Because Hi-C methods require intact cell nuclei, we tested a subset of bird samples from our preservation experiments directly using the Arima-HiC protocol. We tested blood and muscle samples in three different treatments: without preservatives, in EtOH, and using DNAgard. Each preservation method was subjected to three temperature treatments: immediately flash-frozen, 6 hr at 4°C, and one week at room temperature (20–25°C). After temperature treatment, each sample was moved to –80ºC. Blood with no preservative at room temperature for one week was excluded from this set. Two technical replicates of each sample were prepared and sequenced at Arima Genomics following their standard protocol.

FEMTO: A directory containing subfolders with FEMTO Pulse outputs. Files are grouped by run and by species. See "sample info.txt" within each taxon folder for a list of samples and treatments. These files can be opened with a Fragment Analyzer software such as ProSize.

PFGE: A folder containing images from Pulsed Field Gel Electrophoresis. Each gel has two images associated. One image with no text overlay and one with labeling. 

Scripts: A folder containing two scripts written to analyze data for this study.