This document provides guidance on DNA barcoding methods for marine invertebrates. It discusses best practices for tissue preservation, sampling, DNA extraction, PCR, sequencing, and editing for various taxonomic groups. Key recommendations include using ethanol for preservation if less than 5 years old, mtDNA-rich tissues for sampling, and CTAB extraction protocols for certain phyla due to polysaccharides. Taxon-specific primers are often needed for PCR. Multiplex primer cocktails can be used for sequencing multiple samples simultaneously. Care is required when editing due to possible indels and pseudogenes in some groups.

1. Marine Invertebrates
Dirk Steinke
Short Course on DNA Barcoding Methods 29 Nov 2011

2. Preservation
The good
• Ethanol preserved, not older than 5 years
• frozen tissue
• RNA later
The bad
• Ethanol preserved, older than 10 years at room temp
• DMSO (cross-reacts with Ethanol)
• Isopropanol
The ugly
• formalin
• spirit
•…

3. Sampling
mtDNA rich tissue where possible
• muscle tissue (larger animals e.g. fish)
• legs (arthropods)
• tube feet (echinoderms)
The smaller the better!

4. Extraction
Regular kits will do in many cases, but
Echinoderms
Polychaetes
Mollusks
work much better with CTAB
extraction protocols
(binds to polysaccharides that can inhibit PCR)

5. PCR
Taxon-specific primers are key
Check out http://connect.barcodeoflife.net/group/marinebarcoding

6. PCR
Majority works with Taxon specific primers
one primer pair or cocktail needed
- Fish - Crustacea
- Cephalopoda1 - Annelida
- Gastropoda - Cnidaria
- Pycnogonida - Porifera
- Echinodermata
- Brachipoda
Notoriously difficult
- Foraminifera
- Bivalvia2
- Tunicata
1some families have a tandem copy of COI
2two different COI-versions (male/female)

7. Sequencing
Multiplex primer cocktails
5’ 3’
M13-tail
• M13 tail for sequencing multiplex products
• M13 also useful for standard primer pairs
(Folmer-tailed version very successful)

8. Editing
• some groups exhibit indels more frequently (e.g. crustacea, mollusks)
• watch out for pseudogenes (often easy to spot through stop codons)
• proper alignment is crucial
• some symbiotic bacteria can be amplified using universal primers

9. Thank you!