DOI: 10.5061/dryad.000000041
title: Data from: Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing

description: 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.
description: <strong>Sample
 collection. </strong>We collected samples from species representing
 major taxonomic classes of vertebrates, i.e. house mouse (<em>Mus
 musculus</em>), zebra finch (<em>Taeniopygia
 guttata</em>), Kemp’s Ridley sea turtle (<em>Lepidochelys
 kempii</em>), painted turtle (<em>Chrysemys picta</em>),
 American bullfrog (<em>Rana catesbeiana</em>), and zebrafish
 (<em>Danio rerio</em>). 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
 CO<sub>2</sub> 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.  <strong>Preservation
 treatments.</strong> 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. <strong>DNA
 extraction.</strong> 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. <strong>Assessing sample
 fragment size distributions</strong>. 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. <strong>Hi-C library
 preparation and sequencing. </strong>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.<strong> </strong>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.
description: <strong>FEMTO</strong>: 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.
 <strong>PFGE</strong>: 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. 
 <strong>Scripts</strong>: A folder containing two
 scripts written to analyze data for this study.