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REVIEW

Open AccessTissue sampling methods and standards forvertebrate genomics
Pamela BY Wong1,2, Edward O Wiley3, Warren E Johnson4, Oliver A Ryder5, Stephen J O’Brien6, David Haussler7,
Klaus-Peter Koepfli4, Marlys L Houck5, Polina Perelman8, Gabriela Mastromonaco9, Andrew C Bentley3,
Byrappa Venkatesh10, Ya-ping Zhang12,13, Robert W Murphy1,2,12* and G10KCOS11

Abstract
The recent rise in speed and efficiency of new sequencing technologies have facilitated high-throughput
sequencing, assembly and analyses of genomes, advancing ongoing efforts to analyze genetic sequences acrossmajor vertebrate groups. Standardized procedures in acquiring high quality DNA and RNA and establishing cell lines
from target species will facilitate these initiatives. We provide a legal and methodological guide according to fourstandards of acquiring and storing tissue for the Genome 10K Project and similar initiatives as follows: four-star(banked tissue/cell cultures, RNA from multiple types of tissue for transcriptomes, and sufficient flash-frozen tissue
for 1 mg of DNA, all from a single individual); three-star (RNA as above and frozen tissue for 1 mg of DNA); two-star(frozen tissue for at least 700 μg of DNA); and one-star (ethanol-preserved tissue for 700 μg of DNA or less of mixed
quality). At a minimum, all tissues collected for the Genome 10K and other genomic projects should consider eachspecies’ natural history and follow institutional and legal requirements. Associated documentation should detail asmuch information as possible about provenance to ensure representative sampling and subsequent sequencing.
Hopefully, the procedures outlined here will not only encourage success in the Genome 10K Project but also inspire
the adaptation of standards by other genomic projects, including those involving other biota.
Keywords: Genome 10K, Sequencing, Vertebrates, Genomics, Tissue sampling, Tissue storage, Cell line, Tissue
culture, RNA, DNA

Review
Advances in sequencing technology over the last decade[1-3] have made it feasible to acquire a database forgenomes of 10,000 species of vertebrates, analogous to
the Human Genome Project. The Genome 10K Project(G10K), which proposes to catalogue whole-genome
sequences across living mammals, birds, non-avian reptiles, amphibians, and fishes, will reveal the complex
genomic architecture governing the physiology anddevelopment of closely and distantly related species [4].
Documenting the dynamic variation of species in amanner permitting detailed comparative genomic andgenetic analyses will provide invaluable insight into thefundamental principles driving species’ adaptation to* Correspondence: bob.murphy@utoronto.ca
1
Department of Ecology and Evolutionary Biology, University of Toronto, 25
Willcocks St, Toronto, Ontario M5S 3B2, Canada
2
Centre for Biodiversity and Conservation Biology, Royal Ontario Museum,
100 Queen`s Park, Toronto, Ontario M5S 2C6, Canada
Full list of author information is available at the end of the article

ecological and environmental interactions [5-7]. In thisregard, the “Genomics Era” [7] holds promise for new
population-genomic approaches to intraspecific biogeography [e.g., [8,9]] and population genetics [e.g., [10,11]]that are imperative to effective biodiversity conservation
across species [5,6,12-14]. The process of sequencing,assembling, interpreting, and applying information using
whole-genome approaches is starting and quickly building momentum.
As a first step, the G10K Community of Scientists [4]proposes to assemble a collection of tissues and DNA
samples representing 10,000 extant vertebrate species.
This biospecimen collection will be increasingly valuable
the more it is able to standardize procedures for collecting, transporting, and storing high-quality tissue samples.
This process, which applies to all genomics projects, isremarkably complex and daunting, especially because
many of the existing tissue collections have a history ofdevelopment, preservation, and storage for different© 2012 Wong et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

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purposes. Many potential resources are not suitablegiven the requirements of current sequencing andassembly technologies, generally because of insufficientyields of high molecular weight DNA from ethanolpreserved or improperly frozen tissue samples.
Proper collection and preservation of tissues acrossvertebrate species is fundamental to establishing cell cultures and isolating nuclear and mitochondrial DNA,
RNA, and potentially proteomes suitable for genomics,
as these materials are susceptible to rapid post-mortem
degradation or degradation following tissue-samplingfrom living specimens. High-quality DNA will facilitate
de novo assemblies of whole genomes, while viable cell
cultures and RNA will be critical for experimental molecular and cell-based investigations, physical mapping ofgenes onto chromosomes (e.g., fluorescence in situ
hybridization, radiation hybrid mapping, chromosome
flow sorting), transcriptome analyses, and annotation.
The standards of material collected for G10K and otherprojects will vary according to the exigencies of collecting specimens, including ease and method of capture,
availability of specimens, feasibility, tissue type, andtarget quantity and quality. G10K, its contributors, andother researchers will benefit from adopting standardized
methods that correspond to their goals of collection.
Therefore, we propose standards for sample collection tofacilitate and, more importantly, motivate the highest
quality, and most broadly useful and valuable samples
possible. We also review a range of issues related to
selection and documentation of the individual sampled
animals including some pertinent legal and ethicalconsiderations.
To help standardize and assess the quality of tissues
collected, we propose four categories for classifying theutility of tissues and DNA being prepared and reservedfor G10K and similar projects: Four-star (****): sufficient flash-frozen tissue or

immediate extraction of DNA for a minimum of
1 mg of DNA (e.g., enough DNA for at least two
whole-genome sequencing attempts) validated by
DNA barcoding; multiple tissues suitable for RNA
sequencing and transcriptome analysis; and viably
frozen tissue pieces and/or cell lines; Three-star (***): frozen tissue for a minimum of
1 mg of DNA and multiple tissues suitable for RNA
sequencing and transcriptome analysis; Two-star (**): frozen tissue for 700 μg of DNA; and One-star (*): ethanol-preserved tissue for 700 μg
DNA of high or mixed quality (some highly orslightly degraded) and DNA of insufficient quantity(< 700 μg), but of possible value in supplementing
whole-genome sequencing efforts of higher quality
samples.

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These standards have significant implications for thequality and quantity of data for future projects on denovo vertebrate genomics. The following text details
methods for tissue acquisition and preservation in lightof these four standards. At and below the * standard,
attempts at producing whole genome sequences are not
likely to meet with success without reference genomes
and notably greater expense.
A priori considerations

Optimal techniques for acquiring ****, ***, **, and * samples will vary according to species, sex, geographic diversity, and population diversity across the major vertebrategroups [4]. It is critical to consider the individual historyof each specimen in order to maximize a reliable yield oftissue, DNA, and RNA. For some species (e.g., mammals
and birds), blood may be a source of genetic material,
whereas whole specimens may be required to obtain sufficient quantities of DNA in others (e.g., amphibians andnon-avian reptiles). At a minimum, the feasibility of eachprocedure will depend on budget, transport, availability,
health of the source-specimen, and the extent of degradation prior to or after sample collection. Appropriately,
tissue collection should include back-up procedures (e.g.,multiple samples, back-up power supply for freezers,
multiple copies of appropriate documentation) wheneverpossible while minimizing all safety risks, as with any
experimental design.
Apart from sample standards, documentation andarchiving of permits held by every provider is mandatory
for all material collected for G10K. We encourage this forother projects and below consider this universality to be
implicit in all references to G10K. Given difficulties inusing available museum collections, it will be necessary toacquire fresh material, especially for **** and *** samples.
Consequently, all relevant permit and license applications
should be prepared and submitted well in advance of tissue
collection to allow for review and processing time.
Approved written animal care (use) protocols may benecessary from, for example, an Institutional Animal Care
Use Committee (IACUC) or Animal Care Committee(ACC), and in some cases animal health permits will be
required. All procedures must conform to institutional,
local, state, and/or federal guidelines [e.g., [15-19]].
Many countries including the United States are signatories on the “Nagoya Protocol on Access to Genetic
Resources and the Fair and Equitable Sharing of Benefits
Arising from their Utilization to the Convention on Biological Diversity”, promoting genetic research toward conserving biological diversity [[20], see [21] for text].
Accordingly, equal access and benefit-sharing should beensured, in addition to complying with regulations protecting general public health, domestic crops, and livestock orcontrol of trafficking of listed species [e.g., [17,22-25]]. In

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many countries including the United States, Canada,
China, Vietnam, and Mexico, research within nationalparks, nature reserves, marine conservation areas, historicsites or landmarks requires additional research permits(e.g., see [17] for Australia, [26] for USA, [27] for Canada,and [28] for New Zealand). In some cases, research
licenses may apply to particular territories (e.g., Nunavut,
Canada) or cultural permission may be necessary; forexample, consultation with the Maori of New Zealand isrequisite for sampling wildlife that may lead to cultural
sensitivity [29]. It is necessary to acquire permission forsampling natural populations from the appropriate fish,
wildlife, forestry, conservation, and other offices.
Permits and licenses may also be necessary for import
and/or export (e.g., [30]; for a list of permits see [31] forAustralia, [32] for Canada, [33] for New Zealand, and[34] for USA). These constraints can depend on taxon
and country. For example, in Canada, a “Permit to Import Material of Animal or Microbial Origin from Agriculture and Agri-Food Canada” is required for imports
and exports of many but not all vertebrate groups [23].
Prior to air travel, check all potential specimens or chemicals for classification as “Restricted Articles” (may not
be carried as checked or hand-carried baggage on commercial aircraft) by the International Air Transport
Association (IATA) and appropriate “Shippers Declaration for (Non) Dangerous Goods” certificates should beacquired [35]; a list of potentially required documents isavailable [36]. Compliance with applicable regulations
routinely requires declaration of all specimens to customs officials upon arrival after crossing borders. Manylarge laboratories or institutions and museums alreadyhave permits in place and have trained and experienced
specialists; researchers are encouraged to seek their help
and advice to streamline the permit application process.
Collecting tissues for G10K
Description of specimens for G10K

Careful identification and validation by a specialist is necessary to ensure true subsequent sequencing representation. In cases where cryptic species are likely to exist,
sample from the type locality when possible. Species
selected for G10K should have established biologicalrelevance for society and the scientific community. Characteristics include extreme phenotypes, phylogenetic
uniqueness (hence applications for comparative biology),interests for conservation, and relevance for other scientific studies. Targeted species should allow multiple samples to be collected from one individual (e.g., large bodysize) for high-coverage sequencing. Smaller amounts ofDNA (about 30–100 μg) from several individuals of thesame species will support light-coverage sequencing forthe discovery of single-nucleotide polymorphisms. Triads(parent-parent-child or sibling-parent samples) will

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promote further genome exploration and haplotype
description.
At least one reference species will also be targeted for
130 vertebrate orders [4] and these should be included
among the **** collections. These species will supply
high-quality assemblies (thus requiring storage and preservation of more tissue). Reference species will facilitate
the assembly of closely related non-reference species.
Characteristics of reference species include accessibility
to multiple tissues (for transcriptome analysis), samples,
individuals and subspecies, as well as the possibility ofbanking both sexes (assuming chromosomal sex determination). Samples from reference species should also
allow for potential chromosomal mapping (karyotyping);
the establishment and banking of viable frozen cell cultures is encouraged. Consistent with standards fortargeted ordinal representatives, as much information as
possible should be collected for all specimens in order toeffectively link phenotypes of a particular specimen“type” to its genome (see Lodging of vouchers below).
The sequencing of both sexes (e.g., non-recombining
and sex-determining regions) provides important
markers and crucial data for evolutionary and biologicalinferences. In spite of this, few genomic-wide sequences
are now assembled for both sexes as current genome
sequencing and assembly efforts may be challenging withhighly repetitive data characteristic of sex-determining
chromosomes (e.g., Y chromosome in mammals or W
chromosome in birds). If sex determination is associatedwith structurally diverged chromosomes, collections
from specimens of the heterogametic sex will allow
G10K to obtain as much information as possible for eachsex; for example, sequencing the heterogametic sex willallow for the development of sex-specific markers (Y orW) for applications in sex-biased dispersal or gene flow
relevant to population genetics. Alternatively, sequencing
homogametic individuals will be less expensive anddouble the coverage on the (X or Z) chromosome (e.g.,
equivalent to that of autosomes) [37]. In this time ofshort reads and high coverage (> 50 times), the selectionof the heterogametic sex seems preferable as is illustrated by the adequate X-chromosome assembly of themale compared to female ferret (D. Jaffe, personalcommunication).
Freshness

All anaesthetization or euthanasia procedures will requirea priori academic review and should conform to accepted
practices as outlined by the IACUC [38], American
Veterinary Medical Association Guidelines on Euthanasia[39], or requirements specific to other nations. Handlingand use of chloroform, ketamine, pentobarbital, tricaine,
clove oil, or taxon-specific methods must follow legal
procedures [e.g., [15-19]]. Drugs that potentially alter

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RNA expression or DNA quality should be avoided [40].
If euthanasia is necessary, collect tissue as soon as possible. When feasible, the whole carcass should be properly stored as a museum voucher specimen, including theskeleton, for future reference.
We recommend collecting tissue opportunisticallyfrom live or freshly euthanized specimens for **** and ***standards whenever feasible, while minimizing loss ofvalue of the animal for museum preparations. Wildcaught animals from precise geographical locations, especially type localities of species, are preferred. Noninvasive methods and biopsies will be less damaging to
specimens and hence the collected material. Record thegeneral health of all specimens and any obvious parasites. Avoid encysted or parasitized tissues whenever possible. Caution should be exercised when collecting tissue
from cancerous or damaged organs to ensure that
healthy (versus cancerous) tissue is used as the sourcefor genomic sequencing. Diseased and dying individuals
may lead to altered RNA expression, thus affecting
transcriptomes.
Tissue from salvaged dead animals will generally not
be suitable for either assessment of transcriptomes or
the preparation of cell cultures, unless they are exceptionally fresh. These tissues will likely contain only
5–20% of the quality of DNA extractable from fresh tissue [41], resulting in small DNA fragments or high proportions of mitochondrial DNA that are not suitable forthe preparation of large-insert libraries, which are preferable for lower-cost sequencing and high-quality genome
assembly. Because long-term storage usually leads to ahigher likelihood of degradation and contamination, thesampling of sub-surfaces of soft tissues is desirable [42].
The majority of archived museum collections will only
allow for ** or * standards due to limited tissue volume
and storage in ethanol [43] or other preservatives (seePreserving tissues for G10K). Samples collected from dry
mummified specimens are generally not suitable forsequencing and assembly of a de novo genome. However,for species where archived tissue or bone is the only
available source of DNA (e.g., extinct species), G10K has
a special sequencing initiative, where protocols are evaluated on a case-by-case basis (R.E. Green, personal communications). The respective sampling requirements andmethods for these specimens are beyond the scope of
this paper. In any case, all tissue collected from archived
museum collections will require documentation of
approvals and links to the archival institutions and specimen data.
Selection of tissue for quality and quantity

Sterile methods are critical to the efficacy of G10K material (DNA, RNA, and either cell lines or tissue culture),
hence all ****, ***, **, and * collections should be acquired

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in isolation of other samples using sterile equipment andstored separately to avoid contamination. When collecting a whole specimen, dissect and remove the stomach
and intestine to avoid potential contamination of DNA
from consumed food items (however, immediately saveand/or prepare these tissues for transcriptomes, as discussed below). Never mix or combine specimens; eachsample should correspond to a single individual, not a
combination of multiple individuals. In particular, sample
DNA, RNA, and cell cultures from the same individual
whenever possible. Pertinent reagents should be clean andall instruments or containers should be autoclaved prior touse. Ideally, gloves and dissecting equipment should be
disposable and changed between collecting samples. Pay
special attention to avoid cross-contamination by humantissues.
Quality of collected ****, ***, **, and * standards willvary with quantity of DNA and RNA and the ability toestablish viable cell lines, in addition to feasibility. While
one sample of DNA from a particular specimen may suffice regardless of tissue, separate samples from separate
tissue types are desirable for RNA to achieve high coverage of the diverse transcriptome. Tissue should be sufficient for at least 1 mg of DNA (approximately 1 x 1 x
1 cm3) for **** and *** standards. The ** and * standards
require about 700 μg of DNA. Although any soft tissue
may yield good quality (high molecular weight) genomic
DNA, testis provides the highest yield and hence this is
the preferred tissue in species having heterogametic or
temperature-dependent sex determination. For immature
specimens and homogametic individuals (females in the
XY system, males in the WZ system), liver is the next
best tissue. Because bile salt contamination can affect tissue stability [41], avoid the gall bladder when samplingfrom the liver and process the sampled tissue as soon as
possible. Other soft tissues such as brain, kidney, spleen,
heart, and ovary (without eggs) also yield sufficientamounts of DNA but these organs are typically small insize. Liver and other soft tissues (e.g., spleen, pancreas,
lung, glands) are generally prone to faster degradation
due to higher levels of nucleases, thus harder tissues(e.g., muscle, kidney, heart) may be preferable. Thoughskeletal muscle provides large amounts of tissue, yieldsof high molecular weight DNA are small due to thetough nature of muscle fibers.
For large and live specimens, blood is a good source ofhigh molecular weight DNA, and, further, a preferredtissue for constructing large-insert libraries such as bacterial artificial chromosome libraries. Because fishes,
birds, non-avian reptiles, and most amphibians havenucleated red blood cells, blood provides a good sourceof DNA; when red blood cells are non-nucleated, as inmammals and rarely frogs, white blood cells are thesource of DNA [41]. About 3 ml of blood from non-Wong et al. GigaScience 2012, 1:8
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mammalian vertebrates can yield up to 1 mg genomic
DNA and can be easily collected from medium- to largesize specimens. When possible, separate blood cells fromplasma using a centrifuge prior to freezing. Lysing red
blood cells in mammals prior to freezing will yieldcleaner DNA; ideally, use clean buffy coats without
plasma or red blood cells for DNA extractions. Clean(bacterial-free) sperm can also be sampled as additionalsources of DNA using “French straws” [41]. Abdominal
massages, vibrators, or specialized copulatory devices
may allow the collection of ejaculate from at least some
non-avian reptiles [44] and birds [45].
Tissues from multiple organs are preferred for RNA
sequencing. A range of soft tissues can be targeted for
RNA for **** and *** standards (e.g., skeletal muscle,
spleen, heart, blood, kidney, stomach, and other parts ofthe gastrointestinal tract, reproductive organs, liver,brain, eyes, and lung). When applicable, also target
venom and scent glands for RNA. On the one hand,abundant contractile proteins, connective tissue, and collagen in skeletal muscle, heart, and skin tissue may resultin low RNA yield [2]. On the other hand, bone and brain
tissue may be less subject to degradation and thus yield
longer fragments of RNA [42]. When possible, transport
all tissues collected in the field to the lab in either liquid
nitrogen (preferred) or dry ice (where access to liquid
nitrogen may be restricted). However, do not subject tissue intended for cell culture to freezing temperatures
without using a cryoprotectant (see below) [Additional
file 1 and 2].
Tissue cultures and/or cell lines

For tissue cultures (a **** standard), we recommend tissue collected from eyes, though blood and skin canserve as alternatives. For birds, non-avian reptiles, andamphibians, tissue collected from eyes, trachea, gonads,
tongue (amphibians) and blood feathers (birds) arerobust sources for initiating cell cultures. Viably frozen
deep-skin fibroblasts are preferable for most mammals[Additional file 1]; fibroblasts can yield viable cultures
without the need for highly specialized culture media or
conditions. Specimens which are rich in connective tissues, such as mammalian ear punches or tail snips or avian
trachea, yield fibroblast cell lines with a high proliferationlevel. If there is an organ or tissue of specific interest in aparticular species, we recommend collecting viable biopsies of this tissue. Take biopsies using hand-held biopsy
punches (2, 4, 6, or 8 mm diameter), forceps, needle andscissors or scalpel blade, or biopsy darts.
Sampling tissue to establish cell cultures may varyslightly from other collection techniques. Sterility is
especially important to avoid the introduction of bacteria
or fungi, which will inhibit cell growth and prevent
establishment of the culture. Sterile tools are essential,

Page 5 of 12even if all that is available is 70% isopropyl (rubbing)alcohol for cold sterilization. For most specimens, it isbeneficial to wipe down the biopsy area with alcohol
prior to sample collection. In fishes, build-up of mucous
on the skin can lead to an increased chance of contaminated cell cultures, necessitating careful wiping of themucous with sterile gauze prior to sampling [46]. In
mammals, hair and fur can be a major source of contamination. Thus, removal of hair by shaving or clipping thearea followed by cleaning the skin with gauze soaked in
70% isopropanol prior to sample collection will eliminate
or reduce potential contaminants. If shaving is notpossible, a thorough rinse with soap and water followedby rinsing with either 70% ethanol or isopropanol for 15
to 20 seconds is sufficient to reduce surface bacteria or
fungi. Avoid disinfectants such as chlorhexidine solution
because these are too harsh on cells.
Transport biopsies to a laboratory for appropriate
processing in a biosafety cabinet. In the field, working inclose proximity to a burner will also provide a sterile
environment. Ideally, process biopsies for culturing rightaway without freezing and prepare multiple viable seeding stocks. Additional biopsies should be viably frozen asa back-up where cell lines cannot be established on thefirst attempt (e.g., due to contamination). If short-term
storage or transport is necessary, samples from mammals
or other warm-blooded species can be maintained bycompletely immersing in phosphate buffered saline (PBS)or tissue culture medium, such as Dulbecco’s modified
Eagle’s medium (DMEM) or alpha minimum essential
medium (MEM). Supplement this with 1% antimicrobial
and antifungal antibiotics and hold at room temperatureor 4°C. Mammalian skin and ear biopsies stored in tissue
culture medium with antibiotics at 4°C have produced
viable cultures after 3 weeks (ML Houck, unpublishedobservation); tissue samples from birds, non-avian reptiles, and amphibians have also produced viable cell lines
although storage time-tolerance is less than that for
mammals (ML Houck, unpublished observation). Mammalian biopsies can also be stored in CO2 independent
medium supplemented with 7% fetal bovine serum (FBS)
and antibiotics for up to one week. It is possible to transport whole ears (e.g., artiodactyls or carnivores) in cool
conditions without immersion into medium for up to
one week.
For long-term storage requirements, biopsies should
be minced into small pieces (1 mm2) using clean cuts ofa scalpel blade or fine scissors rather than tearing thetissue apart, transferred to vials containing cryopreservation medium (DMEM or alpha MEM supplemented with
1% antibiotic-antimycotic, 10–20% FBS and 10% dimethyl sulfoxide [DMSO]), and frozen in liquid nitrogenor nitrogen vapour within a dry shipper [e.g., Additional
file 2]. It is important to agitate gently the vial to assure

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coating of all pieces with cryopreservative [47]. To
ensure viability, biopsies should be subject to gradualfreezing (1°C per minute) using commercially available
devices (controlled rate freezer, Mr. FrostyW, StratacoolerWetc.) or Styrofoam containers. For field conditions, it ispossible to make a vessel that dips into the neck of a liquid
nitrogen tank to ensure gradual freezing. If gradual freezing is not feasible, vials containing minced biopsies withcryopreservation medium can be plunged directly into a
dry shipper. Lymphocytes from mammalian blood can beisolated and viably frozen using a 10% DMSO solution.
Samples from aquatic species are particularly difficult totransport over long distances or times; recent work withbiopsy samples from fish detects rapid degeneration inany solution at 4°C and inefficient cryopreservation infreezing medium within a dry shipper (G Mastromonaco,
unpublished observation).
Establish cultures following explants or enzymatic
digestion of samples [e.g., Additional file 3]. Enzymatic
digestion typically involves incubation in a collagenase ortrypsin solution for 30 minutes to several hours, depending on the tissue type (e.g., incubate cartilage-derived
biopsies for one day in a collagenase-hyaluronidase solution [48]). Trypsin can be harsh compared to collagenase
thus take care to avoid over-digestion. Explants–where
fibroblasts migrate out of tissue pieces that stick to thebottom of a culture flask–may provide a shorter-lived
cell line than enzymatic digestion [49], which is particularly relevant to small samples. Culture explants ordigested samples using basic cell culture media (e.g.,
DMEM or alpha MEM for most mammals; 1:1 mixture ofalpha MEM and CloneticsTM Fibroblast Growth Medium
for carnivores, elephants, perissodactyls [50] and other“difficult-to-grow” mammals; Liebovitz L-15 for fishes[51], and DMEM or Amniomax C-100 for non-avian
reptiles [52]) supplemented with antibiotics and serum inincubators with 5–6% CO2 at 40°C (birds), 37°C (mammals), 32–33°C (non-avian reptiles), 20–30°C (amphibians), or 15–27°C (fish) [53]. Detailed protocols for cell
culturing are available in Additional file 3, [54], and [55].
For the initial phase of primary culture, antibiotics
should include an antimycotic such as amphotericin B
along with standard penicillin/streptomycin. Gentamycin
can also be used to reduce risk of mycoplasma. Once theprimary culture is established, sole use of penicillin/
streptomycin should be sufficient during passaging.
Avoid the long-term use of strong antibiotics. Pay attention to the temperature of culturing, which should beclose to the body temperature of the animal (e.g., 37°C
for most mammalian cells). For long-term storage, cryopreserve samples at the primary culture stage as well as
the early passage stages using freezing medium as discussed above [56]. All G10K cell lines will be stored inculture and stock centers and cell line centers.

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Lodging of vouchers

All ****, ***, **, and * tissues should be linked to voucher
specimens to ensure integrity of tissue specimen identification and future validation of identity. All tissues andvouchers should be lodged and cataloged within a recognized research collection, along with all necessary
permits (collection, import, export, Convention on International Trade in Endangered Species of Wild Fauna andFlora, Animal and Plant Health Inspection Service, etc.)
and field notes, to ensure positive identification andreproducibility of results. After collecting tissues, vouchers should be prepared either as study skins (mostbirds and mammals) or fixed in a 3.7x buffered formaldehyde solution and then transferred into 70% ethanol
for long-term storage. Large specimens should have formaldehyde injected into body cavities to ensure uniform
fixation of muscle, gut cavity and brain tissue. The skull
and bones of the animal can be useful for morphologicaltaxonomical purposes. Digital images of vouchers taken
while still alive or shortly after euthanasia are importantfor specimen coloration purposes. If maintaining physicalvouchers is not feasible (very large specimens, etc.)
photographic voucher images are acceptable as an alternative as long as positive identification is possible. Access to all samples within their storage location should
be restricted and guarded to avoid disappearance, accidental thawing, and contamination of specimens [57].
All data associated with the specimen should be collected in the field according to the Darwin Core protocols [58] outlining species name, determiner anddetermined date, locality information (country, state,county, locality name, and latitude and longitude), sex,age, size, color patterns and morphs, collector(s), tissue
type (e.g., muscle, liver, blood, etc.), original preservation(e.g., ethanol, liquid nitrogen, etc.), etc. and reported tothe collection in the form of field notes together withany other relevant information (e.g., associated species,
habitat, environmental parameters, etc.). Guidelines forrecording sample information are available [59].
Preserving tissues for G10K

All collected ****, ***, **, and * materials (e.g., tissues,
nucleic acids, cell lines) should be stored in packages
that prevent damage from UV, exposure to light, contamination, and the entry of other chemicals, including
liquid nitrogen. Each package should allow enough room
for tissue expansion during freezing while minimizing air
pockets to prevent drying and degradation. We recommend using sterile Falcon tubes (15 or 20 ml) or plastic
cryotubes with secure screw top lids for collection andsubsequent storage in liquid nitrogen and freezers. Avoid
tubes with pop-off lids; wrap smaller tubes in aluminum
foil or place them into larger tubes [60]. Tissue can be
stored in plastic bags within ultra-cold freezers for

Wong et al. GigaScience 2012, 1:8
http://www.gigasciencejournal.com/content/1/1/8*** and ** standards. Labels should be inside the bag, notwritten on the outside or on a tag; the latter two can belost or obliterated, removing the identity of the sample
and rendering it useless. Plastic bags should not be
stored in liquid nitrogen [60]. In cases of emergency, it ispossible to make aluminum foil packages but fold theminto air-tight packets in advance and transport flat [60].
Seal the tissues by additional wrapping in heavy-duty
aluminum foil prior to storage in liquid nitrogen to avoid
package bursting [60].
Preserve all tissues as soon as possible following collection to eliminate water [40] and minimize oxidative degradation or damage of the genomic materials. Whereas
more time is necessary to degrade or damage nucleic
acids, RNA can degrade rapidly within minutes [2]. For
this reason, all preservation methods should consider
storage time until the target materials are isolated. Effectiveness of preservation, especially critical for RNA
and cell lines, can be enhanced by cutting tissue into
small fragments to increase surface area [2,40,42,43,61,62].
However, excess blending or homogenizing of tissue willlead to further degradation, especially of nuclear DNA[41]. We recommend storage of tissue by immediate freezing. Colder is better. The only exception is fresh biopsy
specimens from which cell cultures are to be immediately
established; for these specimens maintenance at 4°C–not
colder–is appropriate. Secondary methods using preservatives such as ethanol or DMSO will yield varying results[43] and it is preferable to avoid them when possible.
Techniques that involve minimizing desiccation, FTAW
paper, Guthrie cards, vacuum packing, and household
methods [40] are unlikely to work for genome-level
sequencing.
Freezing

Cryopreservation is the most efficient means of preserving genomic material [4,42,57,61,62]. As a **** standard,
flash-freezing in liquid nitrogen halts all chemical andbiological processes that lead to degradation [40]. This
allows for the long-term preservation of viable cells[40,57], and thus DNA, RNA, and proteins [40], provided
a cryoprotectant such as DMSO is used. For cell cultures, optimize cryoprotectants according to tissue-type(as above); concentrations should be high enough to protect cells from crystal formation yet dilute enough to
avoid chemical injury to cells [41]. Similarly, freezing
should allow time for protection from crystal formation
while minimizing chemical damage associated with slow
freezing [41].
We recommend storing samples below −130°C, therecrystallization point of water [40], or as cold as possible within the laboratory to maximize preservation.
Though most expensive to maintain, we recommend
flash-freezing tissue for long-term storage as a ****

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standard. We recommend freezing and storing tissue
below −80°C in the laboratory for ***, **, and * standards
if liquid nitrogen is not available. Tissue degradation canoccur at temperatures between −20 to −80°C [57] andhousehold freezers (−20°C) will be ineffective due totheir defrosting (heating) cycles.
Non-freezing

Transport and maintenance associated with flash-freezing
can be expensive and prohibitively difficult [62,63], potentially limiting **** collections. Ideally, tissues should haveall alcohol drained and be transferred to freezers (−80°C)
or liquid nitrogen as soon as possible after collection toprevent any further degradation. Alternatively, tissue,
blood, and DNA can be stored at room temperature forup to 6 months in DNAgard TissueW [64], DNAgard
BloodW [65], and DNAstableW [66], respectively; these
fluid preservatives may be more convenient in field conditions or during transport to a low-temperature freezer. It
is possible to place tissue samples for RNA in RNAlaterW,
another fluid preservative. Clinical studies show no significant difference in RNA yields between room and lower
temperatures for up to 3 months, though storage above
25°C can limit RNA yields [62]. RNAlaterW is useful for abroad range of tissues and it bears little to no toxicity or
flammability [40]. However, it is quite expensive [e.g.,[67,68]].
Tissue preserved in ethanol will be considered a *standard, as degradation can still occur during DNA extraction [61]. For these collections, use an optimal ethanol concentration of 95–99% to preserve the collected
tissue [40]; it is possible to enhance the effectiveness of
70% ethanol by adding or 1–3% glycerine or 1xTE buffer
instead of water [40]. We recommend adding at least
three times the ethanol to the volume of tissue [61]; ahigher concentration will also be effective [40]. Replacealcohol after 1 to 2 hours to allow diffusion [41] andagain after 2 to 3 days to improve preservation, as tissues
can retain water during this time [40,61]. It is possible toenhance this method of preservation by transferring thetissue to a lysis buffer for 24 hours prior to DNA extraction [40,61].
Shipping

Use couriers for transporting all tissues and specimens
between institutions and from the field. In advance ofshipping, check courier-specific regulations for transporting animal samples. Equipment for flash-freezing
materials may be difficult to access and process[43,62,63] and should be arranged ahead of time. Liquid
nitrogen may be acquired from gas and welding suppliers, universities, hospitals, mining operations, militaryfacilities, and other institutions in vacuum-insulatedtanks or portable dry-shippers (e.g., used in absorbing

Wong et al. GigaScience 2012, 1:8
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spills) [40]. Transport permits are required for liquid nitrogen and dry ice, which classify as “Restricted Articles”.
In both cases, confirm updated regulations prior to shipping [69]. Shipping is usually associated with heavy, nonpressurized metal tanks, though it is possible to checkthese tanks as baggage if they are empty (e.g., during shorttrips), but at the discretion of the aircraft’s pilot [69]. The
packing and insulation of these tanks requires large
volumes of space. It is possible to reduce shipping volumes
by placing smaller samples in an isolated vacuum-insulatedor Styrofoam box of dry ice. Add plastic tubes filled withwater should to tanks with few samples [41].
Though ethanol is inexpensive, accessible at field sites,and does not require extensive precautions for field use[62], it is flammable, evaporates quickly, and classifies asa “Restricted Article”. Recent regulations allow for thetransport of scientific specimens in small quantities of
ethanol (30 ml internal package: 500 ml total) as nondangerous goods: IATA Special Provision A180 for International Shipping and a letter of interpretation from the
Department of Transportation for domestic shipping[35,70,71]. To ship as non-dangerous goods, specimens
and tissues are required to be packaged and marked
according to these provisions. DNAgardW and DNAstableW reagents are not “Restricted Articles”, thus negating
most regulations associated with transport. However, a“Shipper’s Certification of Articles Not Restricted” may
still be required [35,36]. We do not recommend using
other methods of fluid preservation (e.g., lysis buffers,
DMSO) maintained at room temperature for genomic
DNA [42,61], though in some cases these methods may
be effective when combined with freezing [43].

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material. Constructing libraries using degraded DNA leads
to underrepresentation and low rates of insertions. Although the average read lengths of current next-generation
sequencers are short (70–400 bp) and next generation
sequencers may require a minimum of 200 base pairs foreach read [3], it is essential to generate paired-end reads
from libraries with different insert sizes, ranging from100 bp to 150 kb. High molecular weight is particularly
required for preparing large insert-size (20 kb to 40 kb)
libraries whose paired-end reads provide the critical longrange linkage required for a good genome assembly. It is
possible to estimate DNA quality by running 100–200 ng
of DNA on a low-density agarose gel with a high molecular-weight marker (with bands over 20 kb). For example,
samples can run on a 0.6% agarose gel at 70–90 V for 1 to
3 hours with a λ-Hind III Digest ladder. The major DNA
band from the sample should be larger than the 23 kb
band in the ladder. For pulsed-field gel electrophoresis, themain DNA band should be 40 kb or more. DNA purity
Table 1 Tissue standards (italicized) for vertebrate
genomics corresponding to storage and quality of target
materials (bolded)
Four-starThree-star

X

X

Two-star

One-star

DNA quantity
1 mg> 700 μg

X

≤ 700 μg a

X

Target materials
Cell lines/tissue culture

X

RNA

X

X

Identification and quality assessment of DNA and RNA

DNA

X

X

DNA barcoding—where 648 bp on the 5’ end of cytochrome c oxidase subunit I of the mitochondrial regionis sequenced [see [69,72,73], and [74] for more information]—can be a standard, inexpensive, and rapid method
for confirming species and tissue identity, especiallywhen employing current high-throughput approaches. A
standard quality and barcoding [69,72-74] assessment isrequisite prior to sending samples for whole-genome sequencing and is especially important for historic samples
retrieved from museum collections. Thus, G10K verystrongly recommends barcoding as a **** standard.
Whenever possible, it is important to consider levels ofheterozygosity–estimated during the first round of sequencing–because the appropriate approach to genome
sequencing and assembling depends on heterozygosity;
sequencing and assembly are easier with lower heterozygosity. Though anticipated sequencing technologies may
accommodate DNA of lesser quality and quantity, currently available instruments such as the Illumina HiSeq
or Roche 454 require high molecular weight startingSpecimen type c
Xd

X

X

Xb

Salvaged

X

X

Voucher

X

X

Live/freshly euthanized

Storage
RNAlaterWX

DNAgard/DNAstableWX≤ −130°C

X≥ −80°C≥ −20°C
Ethanol

X
X
X

Smaller quantities (30 to 100 μg) from multiple individuals of the same
species will support light-coverage sequencing for single-nucleotide
polymorphism discovery.
b
High-quality or slightly degraded DNA of small quantities will not likely be
sufficient for whole-genome sequencing; these samples may supplement
whole-genome sequencing efforts of higher-quality samples.
cStandards will vary depending on tissue selection and natural history of thespecimen.
dFour-star samples should also include reference species for aligning de novo
sequences of closely related species (see text for more details).
aWong et al. GigaScience 2012, 1:8
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can be subsequently assessed by using standard 260/280,
260/270, and 260/230 ratios. DNA should be free of proteins, polysaccharides, phenol, or other contaminants. It is
possible to estimate the actual amounts of double-stranded
DNA using a fluorometer (e.g., either Qubit fluorometeror Agilent Bioanalyzer); spectrophotometric evaluations
might not provide accurate estimates for double-strandedDNA.
RNA preparations are often contaminated with residual
genomic DNA and should be routinely treated with
RNAase-free DNAse I followed by phenol-chloroform
extraction and ethanol precipitation. The quantity of RNA
can be determined by measuring the optical density at
260 nm in a spectrophotometer (an RNA solution with an
optical density of 1 at 260 nm contains approximately
40 μg RNA per ml). It is possible to verify the quality ofRNA by running approximately 1 μg of total RNA on a
1.2% denaturing agarose gel together with an RNA ladderand ethidium bromide staining. The presence of sharp andbright ribosomal (28 S and 18 S) RNA bands indicatesgood quality. If RNA has been degraded, ribosomal RNA
bands appear fuzzy or smeary; do not use such samples for
preparing messenger RNA. Purity (absence of ribosomal
RNA contamination), quantity, and size distribution ofmessenger RNA can be assayed using lab-on-a-chip technology such as the RNA 6000 LabChipW kit with theAgilent 2100 bioanalyzer. This assay is rapid, requires

minimal amount of samples (25 to 250 ng/μl), and provides very precise estimates.
Finally, it is necessary to deposit all generated
sequences with an open access repository, such as GenBank, and designate the voucher or tissue holding institution along with any publications emanating from theuse of tissues to ensure fidelity and linking of all data to
the original source organism and allow attribution of thecollection. If appropriate, it is important to acknowledge
or list as a middle author “Genome 10K Community of
Scientists” (G10KCOS), as we have done herein, so thatthe community can track the fruits of its work. Sequenceand publication information should be included in therelevant database, which ideally should be accessible
online.

Conclusions
The G10K project will attempt to follow the standards
outlined herein (Table 1; Figure 1): **** (tissue stored inliquid nitrogen for at least 1 mg of DNA, isolated RNA
and cell line/tissue cultures), *** (frozen tissue for at least
1 mg of DNA and isolated RNA), ** (frozen tissue for700 μg DNA), and * (tissue preserved in ethanol for lessthan 700 μg DNA). We strongly encourage other vertebrate genomic initiatives to adopt this standard. Regardless of standard, it is imperative that all samples
collected for G10K follow relevant legal requirements

Reference species
Natural history

Species selection

IACUC / ACC review

Cultural permission

Sampling

Animal health

Geography
Storage
Protected areas

Import / Export
Transport
Federal species protection
CITES

DNA / RNA isolation andquality assessment

Establishment of cultures

Lodging of vouchers

Preservation

Figure 1 Considerations (dotted), potential permit requirements (gray) and procedures (bold) in acquiring Genome 10 K materials(highlighted). Document as much information as possible on permits, sampling events, species identification, and voucher locations.

Wong et al. GigaScience 2012, 1:8
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and regulations for their acquisition. The **** standard oftissue collection and preservation is preferred and thiswill likely require the acquisition of new materials. In
contrast, the * standard may not be suitable for genomiclevel sequencing given current technological constraints.
However, * collections from rare species where sampling
may be difficult will still be useful for initial wholegenome sequencing attempts. These guidelines can also
be extended to projects focusing on invertebrates (e.g.,i5K [75]), plants, and fungi through similar permit, transport, and storage procedures, and particularly considerations where species identification is difficult (e.g.,barcoding and archiving procedures). However, someethical considerations may not be relevant (e.g., animal
use protocols in invertebrates are restricted to cephalopods) and specialized protocols for tissue collection (e.g.,
animals with smaller body sizes) and establishment of
viable cultures (e.g., plants) may differ. Accordingly,
quantity and quality standards should be established in asimilar fashion (e.g. *, **, ***, and ****) at least for invertebrate, plant, and fungi groups. We hope that the methods and procedures discussed herein will not only foster
initiatives toward the G10K project, but also contributeto a synchronized understanding of the genetic processes
heretofore not available.

Additional files
Additional file 1: A sample mammalian skin biopsy procedure forsubsequent cell culturing.
Additional file 2: A sample protocol for freezing tissue biopsy samples
prior to subsequent initiation of cell culture.
Additional file 3: A sample protocol for the preparation of primarycultures using collagenase digestion.
Abbreviations
G10K: (Genome 10K); ****: (Four-star); ***: (Three-star); **: (Two-star);*: (One-star); ACC: (Animal Care Committee); IACUC: (Institutional Animal Care
Use Committee); IATA: (International Air Transport Association);
PBS: (phosphate buffered saline); DMEM: (Dulbecco’s modified Eagle’s
medium); MEM: (minimum essential medium); FBS: (fetal bovine serum);
DMSO: (dimethyl sulfoxide); G10KCOS: (Genome 10K Community of
Scientists).
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
This research was supported by a Natural Sciences and Research Council of
Canada Discovery Grant 3148 and by a Visiting Professorship for Senior
International Scientists from the Chinese Academy of Sciences to RWM. Thiseffort was also supported by the National Science Foundation Assembling
the Tree of Life Grant DEB0732819 to EOW. The authors would like to thankR.E. Green and B. Shapiro for providing insight into G10K’s sequencing
initiative for extinct species. This project grew out of discussions by G10KCOS
at the second G10K meeting held in Santa Cruz, California on 14–18 March,
2011.
Author details
Department of Ecology and Evolutionary Biology, University of Toronto, 25
Willcocks St., Toronto, Ontario M5S 3B2, Canada. 2Centre for Biodiversity and1Page 10 of 12

Conservation Biology, Royal Ontario Museum, 100 Queen`s Park, Toronto,
Ontario M5S 2C6, Canada. 3Division of Ichthyology, Biodiversity Institute,
University of Kansas, 1345 Jayhawk Boulevard, Lawrence, Kansas 66045, USA.
4
Laboratory of Genomic Diversity, National Cancer Institute, 31 Center Drive,
Frederick, Maryland 21702-1201, USA. 5San Diego Zoo Institute forConservation Research, 15600 San Pasqual Valley Road, Escondido, California
92027, USA. 6Theodosius Dobzhansky Center for Genome Bioinformatics, St.
Petersburg State University, 8 Viborgskaya Street, St. Petersburg 194044,
Russia. 7Center for Biomolecular Science and Engineering, University ofCalifornia Santa Cruz, 1156 High Street, Santa Cruz, California 95064, USA.
8
Laboratory of Animal Cytogenetics, Institute of Molecular and Cellular
Biology, Lavrentiev 10, Novosibirsk 630090, Russia. 9Reproductive Physiology,
Toronto Zoo, 361A Old Finch Avenue, Scarborough, Ontario M1B 5K7,
Canada. 10Comparative Genomics Laboratory, Institute of Molecular and Cell
Biology, A*STAR, Biopolis, Singapore 138673, Singapore. 11Genome 10K
Community of Scientists. http://www.genome10k.org/, Santa Cruz, California ,USA. 12State Key Laboratory of Genetic Resource and Evolution, Kunming
Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu,
Kunming, Yunnan 650223, China. 13Laboratory for Conservation andUtilization of Bioresources, Yunnan University, 2 North Cuihu Road, Kunming,
Yunnan 650091, China.
Authors’ contributions
RWM and EOW conceived the project. PBYW and RWM structured the draft
and provided final editing. PBYW coordinated and drafted the manuscript,
and synthesized comments provided by all authors. All authors contributed
critically important comments. EOW, OAR, WEJ, and RWM contributed toapplications in ichthyology, mammalogy, ornithology, and herpetology,
respectively. WEJ, SOB, DH, PP, YPZ, and BV contributed to constraints on
sequencing genomes. BV contributed to techniques of DNA and RNA
extraction and storage. ACB contributed legal and ethical requirements forbiospecimen transport. PP and GM provided protocols for tissue acquisitionand storage for the establishment of cell lines and tissue cultures; OAR andMH provided protocols in Appendices 1 and 2. All authors read andapproved the final manuscript.
Authors’ information
The G10K project has been coordinated by OAR, SJO, and DH: OAR and SJO
are also General Policy Group and Mammal Group members and DH is also a
General Policy Group and Analysis Group member. YPZ is a General PolicyGroup and Mammal Group member. WEJ is Committee Chair for theMammal Group, and EOW and BV are Committee Co-Chairs for the FishGroup. BV is the Research Director of the Comparative Genomics Laboratory
at the Institute of Molecular and Cell Biology, A*STAR, Singapore. RWM is a
Committee Co-Chair for both the Amphibian Group and the Non-avian
Reptile Group. KPK is a Cancer Research Training Award Fellow at the
National Cancer Institute-Frederick and Manager of the G10K database.
Received: 8 March 2012 Accepted: 12 July 2012
Published: 12 July 2012
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doi:10.1186/2047-217X-1-8
Cite this article as: Wong et al.: Tissue sampling methods and standards
for vertebrate genomics. GigaScience 2012 1:8.

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