Nov 22, 2021
ESTABLISHMENT OF A SPECIMEN/TISSUE BANK AND ASSOCIATED DNA REFERENCE DATA FOR eDNA ANALYSIS
- Luca Mirimin1,
- Dulaney Miller1,
- Sara Fernandez1
- 1Galway Mayo Institute of Technology
Protocol Citation: Luca Mirimin, Dulaney Miller, Sara Fernandez 2021. ESTABLISHMENT OF A SPECIMEN/TISSUE BANK AND ASSOCIATED DNA REFERENCE DATA FOR eDNA ANALYSIS. protocols.io https://dx.doi.org/10.17504/protocols.io.bzn2p5ge
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 01, 2021
Last Modified: November 22, 2021
Protocol Integer ID: 54714
Funders Acknowledgements:
EMFF/MI
Grant ID: SERV-19-MEFS-004
Abstract
This protocol is intended to provide guidelines on the curation and establishment of a specimen/tissue bank and associated DNA sequence data to be used as reference material/data for subsequent environmental DNA (eDNA) analysis, with particular emphasis on marine non-indigenous and invasive species.
Guidelines
This documents provides recommendations on the processing of marine samples (mixed or individual) for the collection, preservation and taxonomic identification of non-indigenous and invasive marine species.
Materials
SAMPLE COLLECTION
Materials will depend on the sampling method.
However, ensure that user safety and biosecurity are always taken into account prior to conducting field work.
SAMPLE PROCESSING (TISSUE BANK)
Ethanol AbsoluteLennoxCatalog #CRTSE0021612
Formalin solution neutral buffered 10%Sigma AldrichCatalog #HT501128-4L
Disposable gloves
Labcoats
Eye protection equipment
2mL screw cap tubes
Pencil
Paper labels (preferably waterproof)
Tweezers
Scalpels
Scissors
Microscope (compound and/or dissecting)
Photo camera
Buckets
Sieve (1mm)
Filter membranes (10um)
Vacuum pump
Virkon aquaticAgriDirect
DNA EXTRACTION
Molecular laboratory equipped with:
Micropipettes
Heating block or oven
Microcentrifuge
DNA extraction kit of choice (e.g. DNeasy Blood & Tissue Kit (QIAGEN)QiagenCatalog #69504 , or E.Z.N.A.® Mollusc DNA Kit (Omega Bio-tek)Omega BiotekCatalog #D3373-01 )
MOLECULAR BARCODING
Molecular laboratory equipped with:
Micropipettes
PCR reagents of choiceed (e.g.Platinum II Taq Hot-Start DNA Polymerase (Invitrogen)Thermo Fisher ScientificCatalog #14966001 or GoTaq® DNA Polymerase (Promega)PromegaCatalog #M3001 )
Thermal Cycler (PCR)
e.g.
Equipment
SimpliAmp Thermal Cycler
NAME
PCR
TYPE
Applied Biosystems
BRAND
A24811
SKU
LINK
Any standard PCR thermocycler will suffice
SPECIFICATIONS
Gel electrophoresis apparatus
Agarose gel
TAE bufferTAE (Tris Acetate-EDTA) BufferCatalog #T6025
Standard size ladder (100-1000bp) (e.g. 100 bp DNA Ladder - 500 gel lanesNew England BiolabsCatalog #N3231L )
Protocol materials
Ethanol AbsoluteLennoxCatalog #CRTSE0021612
100 bp DNA Ladder - 500 gel lanesNew England BiolabsCatalog #N3231L
TAE (Tris Acetate-EDTA) BufferCatalog #T6025
Formalin solution neutral buffered 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #HT501128-4L
Virkon aquaticAgriDirect
DNeasy Blood & Tissue Kit (QIAGEN)QiagenCatalog #69504
E.Z.N.A.® Mollusc DNA Kit (Omega Bio-tek)Omega BiotekCatalog #D3373-01
Platinum II Taq Hot-Start DNA Polymerase (Invitrogen)Thermo Fisher ScientificCatalog #14966001
GoTaq® DNA Polymerase (Promega)PromegaCatalog #M3001
E.Z.N.A.® Mollusc DNA Kit (Omega Bio-tek)Omega BiotekCatalog #D3373-01
DNeasy Blood & Tissue Kit (QIAGEN)QiagenCatalog #69504
Ethanol AbsoluteLennoxCatalog #CRTSE0021612
Formalin solution neutral buffered 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #HT501128-4L
Safety warnings
Make sure to establish a Risk Assessment to mitigate any adverse effect on users while carrying out any of these protocols. Useful information (e.g. Safety Data Sheets) should be sourced from the relevant suppliers who procured the materials.
Before start
To minimize risk of cross-sample contamination, note that all re-usable materials (e.g. scalpels, scissors, tweezers) should be decontaminated to remove any traces of DNA. It is highly recommended to carry out these protocols in dedicated and PCR-free laboratories/rooms.
Ensure that each protocol and list of materials is checked before starting any of the procedures. Specifically, make sure that all key materials(e.g. kits) have not been modified or discontinued by the relevant supplier.
Introduction
Introduction
This document includes the following sections:
- A graphical representation of the overall process
- Collection of specimens and associated metadata
- Specimen and tissue bank
- Isolation of genomic DNA
- Molecular barcoding
- Acknowledgements
Graphical overview
Graphical overview
General workflow overview for the establishment and curation of a specimen and DNA reference database
Collection of specimens and associated metadata
Collection of specimens and associated metadata
Specimens can be collected following targeted surveys or opportunistically, using rapid methods (collection by hand, dredges, corers, traps, nets, etc.) or dedicated in situ approaches (e.g. SETL settlement plates; https://www.gimaris.com/Projects/SETL-project). When possible, whole specimens should be preserved for future downstream taxonomic identification.
As for metadata, the minimum information would include location (ideally with exact coordinates), date of collection, method of collection, photographs, and name/contact of person who collected the specimen. For an extensive list of recommended metadata, we recommend consulting guidelines provided in Rimet et al. (2021).
CITATION
Specimen and tissue bank
Specimen and tissue bank
For samples containing multiple organisms (e.g. dredge, SETL), specimens should be sorted and separated into single species groups. If the goal of the survey includes small- to micro-organisms, any small (>1mm) organism should be collected by rinsing with clear seawater (or artificial seawater) any substrate or specimen through a 1mm sieve, whereas the flow-through water should be inspected with a microscope for the presence of micro-organism.
Safety information
To avoid unintentional spreading of non-indigenous species and/or pathogens, make sure to treat any materials and liquids with appropriate disinfectants before disposal (e.g. Virkon aquaticAgriDirect ).
A unique specimen identifier code should be allocated to each specimen collected. The format and style of such code will depend on the intended database/repository.
Preservation conditions will depend on the organism and can vary substantially, however such conditions should be chosen with the purpose of (i) limit DNA degradation during storage and (ii) ensure that key taxonomic features are retained for subsequent identification. However, in the context of marine non-indigenous species, many taxa (including most invertebrates) can be preserved frozen (-20°C to -80°C), using 70-100% Ethanol AbsoluteLennoxCatalog #CRTSE0021612 (for subsequent DNA analysis, but may lead to discoloration affecting taxonomic ID) and/or Formalin solution neutral buffered 10%Sigma AldrichCatalog #HT501128-4L (for subsequent taxonomic identification, but not recommended or DNA isolation).
WARNING: caution should be exercised when handling ethanol and formalin. Please consult the relevant Safety Data Sheets for further information.
Safety information
H225 | Highly flammable liquid and vapour
H319 | Causes serious eye irritation
H317 | May cause an allergic skin reaction
H351 | Suspected of causing cancer
Isolation of genomic DNA
Isolation of genomic DNA
DNA isolation protocols should be adapted to each organisms following the manufacturer's recommendations. However, in the context of marine non-indigenous species, two DNA isolation kits have been used with highly successful rates, including the E.Z.N.A.® Mollusc DNA Kit (Omega Bio-tek)Omega BiotekCatalog #D3373-01 (particularly successful on mollusks and arthropods) and theDNeasy Blood & Tissue Kit (QIAGEN)QiagenCatalog #69504 (for most other animal species, algae and microorganisms).
Subsequent to DNA isolation, template DNA should be quantified using a Qubit Fluorometer (Invitrogen), whereas quality of extracts should be assessed using a small-volume spectrophotometer (Thermo Scientific™).
Equipment
Invitrogen™ Qubit™ 3 Fluorometer
NAME
Accurately measures DNA, RNA, and protein using the highly sensitive fluorescence-based Qubit quantitation assays
TYPE
Invitrogen™ Q33216
BRAND
Q33216
SKU
LINK
Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
LINK
Sample Volume (Metric): Minimum 1µL; Spectral Bandwidth: ≤1.8 nm (FWHM at Hg 254 nm); System Requirements: Windows™ 8.1 and 10, 64 bit; Voltage: 12 V (DC); Wavelength Range: 190–850 nm
SPECIFICATIONS
Molecular barcoding
Molecular barcoding
Note
The choice of genetic markers to be used in molecular barcoding depends on the target organism as well as on the intended taxonomic resolution of the barcode. Here we provide a range of molecular markers and protocols that were successfully used to barcode a wide range of marine non-indigenous species.
| A | B | C | D | E | F | G | H | I | J | K | L | |
| Assay # | Target Taxa | Target Gene | Amplicon Length Range (bp) | Forward Primer Name | Forward Primer Sequence (5'-3') | Forward Primer Source Reference | Reverse Primer Name | Reverse Primer Sequence (5'-3') | Reverse Primer Source Reference | |||
| 1 | Marine metazoans | COI | 658 | LoboF1 | KBTCHACAAAYCAYAARGAYATHGG | Lobo et al. 2013 | LoboR1 | TAAACYTCWGGRTGWCCRAARAAYCA | Lobo et al. 2013 | |||
| 2 | Marine metazoans | COI | 313 | mICOIintF‐XT | GGWACWRGWTGRACWNTNTAYCCYCC | Leray et al 2013 | jgHCO2198a | TANACYTCNGGRTGNCCRAARAAYCA | Leray et al 2013 | |||
| 3 | Marine metazoans | COI | 710 | jgLCO1490 | TITCIACIAAYCAYAARGAYATTGG | Geller et al 2013 | jgHCO2198b | TAIACYTCIGGRTGICCRAARAAYCA | Geller et al 2013 | |||
| 4 | Marine metazoans | 16S rRNA | 567 | 16sar-L | CGCCTGTTTATCAAAAACAT | Palumbi et al 2002 | 16sbr-H | CCGGTCTGAACTCAGATCACGT | Palumbi et al 2002 | |||
| 5 | (Freshwater) Diatoms | RuBisCO | 312 | Diat_rbcL_708F | AGGTGAARYWAAAGGTTCWTAYTTAAA | Vasselon et al 2017 | Diat_rbcL_R3 | CCTTCTAATTTACCWACWACTG | Vasselon et al 2017 |
Note
The following PCR conditions are specific to the selected markers reported above and were optimized using Platinum II Taq Hot-Start DNA Polymerase (Invitrogen)Thermo Fisher ScientificCatalog #14966001 or GoTaq® DNA Polymerase (Promega)PromegaCatalog #M3001 . While the reported reagents and conditions were successful on a range of taxonomic groups, further optimization may be required for specimens that fail to amplify.
Assay 1 (COI - Lobo et al 2013)
CITATION
| A | B | C | |
| Reagent name and starting concentration | Amount per reaction (µL) | Final concentration (per reaction) | |
| Moleculr grade water | to 10 μL | - | |
| 5X Platinum™ II PCR Buffer | 2 | 1X | |
| 10 mM dNTP mix | 0.2 | 0.2 mM each | |
| 10 μM forward primer | 0.2 | 0.2 μM | |
| 10 μM reverse primer | 0.2 | 0.2 μM | |
| Platinum™ II Taq Hot-Start DNA Polymerase | 0.08 | 0.04 U/μL | |
| Template DNA | 1-2 | <200 ng/rxn | |
| Total | 10 |
PCR reagents and concentrations for Assay 1
Thermal cycling conditions:
94ºC-1min
94ºC-30secs
45ºC-60secs x5
72ºC-60secs
94ºC-30secs
54ºC-90secs x45
72ºC-60secs
4ºC-+∞
Assay 2 (COI - Leray et al 2013)
CITATION
| A | B | C | D | |
| Reagent name and starting concentration | Amount per reaction (µL) | Final concentration (per reaction) | ||
| Moleculr grade water | to 20µL | - | ||
| 5X GoTaq PCR Buffer | 4 | 1X | ||
| 10 mM dNTP mix | 0.4 | 0.2 mM each | ||
| 10 μM forward primer | 1 | 0.5 µM | ||
| 10 μM reverse primer | 1 | 0.5 μM | ||
| GoTaq DNA Polymerase | 0.15 | 0.04 U/μL | ||
| Template DNA | 2 | <200 ng/rxn | ||
| Total | 20 |
Thermal cycling conditions:
95ºC-1min
95ºC-15secs
46ºC-15secs x40
72ºC-10secs
72ºC-3min
4ºC-+∞
Assay 3 (COI - Geller et al 2013)
CITATION
| A | B | C | D | |
| Reagent name and starting concentration | Amount per reaction (µL) | Final concentration (per reaction) | ||
| Moleculr grade water | to 20µL | - | ||
| 5X GoTaq PCR Buffer | 4 | 1X | ||
| 10 mM dNTP mix | 0.4 | 0.2 mM each | ||
| 10 μM forward primer | 1 | 0.5 µM | ||
| 10 μM reverse primer | 1 | 0.5 μM | ||
| GoTaq DNA Polymerase | 0.15 | 0.04 U/μL | ||
| Template DNA | 2 | <200 ng/rxn | ||
| Total | 20 |
Thermal cycling conditions:
95ºC-5min
95ºC-1min
48ºC-1min x40
72ºC-1min
72ºC-5min
4ºC-+∞
Assay 4 (16S rRNA - Palumbi et al 2002)
CITATION
| A | B | C | D | |
| Reagent name and starting concentration | Amount per reaction (µL) | Final concentration (per reaction) | ||
| Moleculr grade water | to 20µL | - | ||
| 5X GoTaq PCR Buffer | 4 | 1X | ||
| 10 mM dNTP mix | 0.4 | 0.2 mM each | ||
| 10 μM forward primer | 1 | 0.5 µM | ||
| 10 μM reverse primer | 1 | 0.5 μM | ||
| GoTaq DNA Polymerase | 0.15 | 0.04 U/μL | ||
| Template DNA | 2 | <200 ng/rxn | ||
| Total | 20 |
Thermal cycling conditions:
95ºC-5min
94ºC-1min
55ºC-1min x40
72ºC-2min
72ºC-7min
4ºC-+∞
Assay 5 (RuBisCO - Vasselon et al 2017)
CITATION
| A | B | C | D | |
| Reagent name and starting concentration | Amount per reaction (µL) | Final concentration (per reaction) | ||
| Moleculr grade water | to 20µL | - | ||
| 5X GoTaq PCR Buffer | 4 | 1X | ||
| 10 mM dNTP mix | 0.4 | 0.2 mM each | ||
| 10 μM forward primer | 1 | 0.5 µM | ||
| 10 μM reverse primer | 1 | 0.5 μM | ||
| GoTaq DNA Polymerase | 0.15 | 0.04 U/μL | ||
| Template DNA | 2 | <200 ng/rxn | ||
| Total | 20 |
Thermal cycling conditions:
94ºC-1min
95ºC-30secs
46ºC-30secs x40
72ºC-30secs
72ºC-10min
4ºC-+∞
Successful amplification and confirmation of expected size of PCR products should be carried out by agarose gel electrophoresis.
(Optional) A PCR clean-up step can be included at this stage, but in many cases it is not necessary.
DNA sequences should be obtained by Sanger sequencing using the Forward and/or reverse primers.
Protocol
NAME
Sanger SequencingCREATED BY
Lívia Sacchetto
Citations
Step 3
Rimet F, Aylagas E, Borja A, Bouchez A, Canino A, Chauvin C, Chonova T, Ciampor Jr F, Costa FO, Ferrari BJD, Gastineau R, Goulon C, Gugger M, Holzmann M, Jahn R, Kahlert M, Kusber W-H, Laplace-Treyture C, Leese F, Leliaert F, Mann DG, Marchand F, Méléder V, Pawlowski J, Rasconi S, Rivera S, Rougerie R, Schweizer M, Trobajo R, Vasselon V, Vivien R, Weigand A, Witkowski A, Zimmermann J, Ekrem T. Metadata standards and practical guidelines for specimen and DNA curation when building barcode reference libraries for aquatic life
https://doi.org/10.3897/mbmg.5.58056Step 8
Leray M, Yang JY, Meyer CP, Mills SC, Agudelo N, Ranwez V, Boehm JT, Machida RJ. A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents.
https://doi.org/10.1186/1742-9994-10-34Step 8
Geller J, Meyer C, Parker M, Hawk H. Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys.
https://doi.org/10.1111/1755-0998.12138Step 8
Palumbi S, Martin A, Romano S, McMillan W, Stice L, Grabowski G.. The Simple Fool's Guide to PCR, Version 2.0
Step 8
Vasselon, V., Rimet, F., Tapolczai, K., & Bouchez, A.. Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte Island, France)
https://doi.org/10.1016/j.ecolind.2017.06.024Step 8
Lobo, J., Costa, P.M., Teixeira, M.A., Ferreira, M.S.G., Costa, M.H., Costa, F.O.. Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans
https://doi.org/10.1186/1472-6785-13-34