ESTABLISHMENT OF A SPECIMEN/TISSUE BANK AND ASSOCIATED DNA REFERENCE DATA FOR eDNA ANALYSIS

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

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).

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.

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.

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™).

Equipment

Value	Label
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

Equipment

Value	Label
NanoDrop™ One UV-Vis Spectrophotometer	NAME
spectrophotometer	TYPE
Thermo Scientific	BRAND
ND-ONE-W	SKU
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

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

Assay 1 (COI - Lobo et al 2013)

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)

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)

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)

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)

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.

Sanger Sequencing

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 GenBank and BOLD BOLD is strongly encouraged.