TITLE: ESTABLISHMENT OF A SPECIMEN/TISSUE BANK AND ASSOCIATED DNA REFERENCE DATA FOR eDNA ANALYSIS
AUTHORS: Luca    Mirimin, Dulaney Miller, Sara Fernandez

[DESCRIPTION]
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.

[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.

[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.

[STEPS]
SECTION: Collection of specimens and associated metadata
3. 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).
 

SECTION: Specimen and tissue bank
4. 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.
 

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.
SECTION: Introduction
1. 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

SECTION: Isolation of genomic DNA
6. 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   (particularly successful on mollusks and arthropods) and the   (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™).
 

 

SECTION: Molecular barcoding
8. 
 



Assay 1 (COI - Lobo et al 2013)
 

 


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)


 


 
 


Thermal cycling conditions:

95ºC-1min

       95ºC-15secs
       46ºC-15secs             x40
       72ºC-10secs

       72ºC-3min

4ºC-+∞

 
 
 Assay 3 (COI - Geller et al 2013)

 

 

Thermal cycling conditions:

95ºC-5min

       95ºC-1min
       48ºC-1min             x40
       72ºC-1min

       72ºC-5min

4ºC-+∞
 
 
 Assay 4 (16S rRNA - Palumbi et al 2002)

 
 

 
 


Thermal cycling conditions:


 95ºC-5min

       94ºC-1min
       55ºC-1min             x40
       72ºC-2min

       72ºC-7min

4ºC-+∞
 


Assay 5 (RuBisCO - Vasselon et al 2017)


 

 
 


Thermal cycling conditions:

 
 94ºC-1min

       95ºC-30secs
       46ºC-30secs             x40
       72ºC-30secs

       72ºC-10min

4ºC-+∞
SECTION: Specimen and tissue bank
5. 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%  (for subsequent DNA analysis, but may lead to discoloration affecting taxonomic ID) and/or   (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.
 

SECTION: Graphical overview
2.   

SECTION: Molecular barcoding
9. 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.
 

Raw DNA sequence data should be inspected using any suitable software (e.g. MEGA, Geneious) and curated to ensure high quality (error-free) of final data.

Linking DNA sequence data to public repositories such as GenBank and BOLD is strongly encouraged. 
SECTION: Molecular barcoding
7.