TITLE: MBA DTOL - DNA extraction and barcoding of Marine Metazoans 
AUTHORS: Rebekka Uhl

[DESCRIPTION]
This document provides a guide to the protocols used by the Marine Biological Association for DNA extraction and barcoding of marine metazoans sampled for the Darwin Tree of Life project. The selection of organisms described are due to these species requiring more optimized techniques than the high-throughput method otherwise used by the Natural History Museum (dx.doi.org/10.17504/protocols.io.8epv5x7qjg1b/v1). Please note that this is a working document, with more organism-specific barcoding methods being added as they become optimized. The methods described are generally successful for the selected groups, but may still require further optimization for some samples. 


[STEPS]
SECTION: DNA Barcoding: Ascidians
2. Reference: Adapted from Salonna et al., 2021 
Salonna, M., Gasparini, F., Huchon, D., Montesanto, F., Haddas-Sasson, M., Ekins, M., McNamara, M., Mastrototaro, F. and Gissi, C., 2021. An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode. Scientific reports, 11(1), p.4078.

Primers: COI barcode
     F      dinF      CGTTGRTTTATRTCTACWAATCATAARGA        R      Nux1R      GCAGTAAAATAWGCTCGRGARTC   

 PCR reaction mix: 
     Mastermix      1x        GoTaq 5x buffer      10ul        MgCl2 (25mM)      4ul        dNTPs (10uM each)      1ul        Primer (10uM)      2ul (each)        Mol. H2O      29.75ul        Taq      0.25ul        DNA template      1ul (usually 1:10 dil. of
  extracts)         Total      50ul    

Thermocycling conditions: 
95C, 3mins, 30x[95C, 30s; 50C, 30s; 72C, 1.5, mins], 72C, 5mins

If unsuccessful, try repeating PCR with x35 cycles instead (generally required) 
SECTION: DNA barcoding: Fish
3. Reference: Adapted from Moran et al., 2016
Moran, Z., Orth, D.J., Schmitt, J.D., Hallerman, E.M. and Aguilar, R., 2016. Effectiveness of DNA barcoding for identifying piscine prey items in stomach contents of piscivorous catfishes. Environmental Biology of Fishes, 99, pp.161-167.

Primer: COI barcode
     F      FishF2_t1      TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC        R      FishR2_t1      CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA   

PCR reaction mix 
     Mastermix      1x        GoTaq 5x buffer      5ul        MgCl2 (25mM)      3ul        dNTPs (10uM each)      0.625ul        Primer (10uM)      0.25ul (each)        Mol. H2O      14ul        Taq      0.125ul        DNA template      2ul         Total      25ul    

Thermocycling conditions: 
94C, 2mins, 35x[94C, 30s; 52C, 40s; 72C, 1min], 72C, 10min 
SECTION: DNA barcoding: Hydroids
4. References: Adapted from Beckman et al., 2023 and Cunningham & Buss, 1993 

Beckmann, L.M., Soto-Angel, J.J., Hosia, A. and Martell, L., 2023. Odd family reunion: DNA barcoding reveals unexpected relationship between three hydrozoan species. PeerJ, 11, p.e15118.

Cunningham, C.W. and Buss, L.W., 1993. Molecular evidence for multiple episodes of paedomorphosis in the family Hydractiniidae. Biochemical Systematics and Ecology, 21(1), pp.57-69.

DNA extraction: 
Left in heat block for 1.5hrs (exoskeleton remains in tube – does not mean DNA is not extracted!) 
Relatively varied DNA concentrations - usually required 1:10 dilution (if >10ng/ul) to reduce contamination. 

Primers: 16s barcode
     F      SHA      ACGGAATGAACTCAAATCATGT      600bp        R      SHB      TCGACTGTTTACCAAAAACATA      600bp   

PCR reaction mix: 
     Mastermix      1x        RedTaq with MgCl2       25ul        Primer (10uM)      1ul        Mol. H2O      22ul        DNA template      1ul (usually 1:10 dil. of
  extracts)         Total      50ul    

Thermocycling conditions: 
94C,5mins, 35x[94C, 30s; 50C, 30s; 72C, 1mins], 72C, 7 mins

If failed, increase reaction above to x39 cycles (some may better with x35 some better with x39 due to extent of contaminants) 
SECTION: DNA barcoding: Barnacles*
5. *Note: This method has been optimized for barnacle barcoding, but may also be useful for other crustaceans as universal COI primers are used (this has not been trialled). 

Reference: Adapted from Benny et al., 2007 

Chan, B.K., Tsang, L.M. and Chu, K.H., 2007. Morphological and genetic differentiation of the acorn barnacle Tetraclita squamosa (Crustacea, Cirripedia) in East Asia and description of a new species of Tetraclita. Zoologica Scripta, 36(1), pp.79-91.

Primer: COI barcodes  
     F      LCO1409      GGTCAACAAATCATAAAGATATTGG      600bp        R      HCO2198      TAAACTTCAGGGTGACCAAAAAATCA      600bp   

PCR reaction mix: 
     Mastermix      1x        GoTaq 5x buffer      10ul        MgCl2 (25mM)      4ul        dNTPs (10uM each)      1ul        Primer (10uM)      1ul (each)        Mol. H2O      31.7ul        Taq      0.3ul        DNA template      1ul         Total      50ul    

Thermocycling conditions: 
94C,3mins, 33x[94C, 30s; 47C, 30s; 72C, 40s], 72C, 3 mins
SECTION: DNA barcoding: Copepods
6. References: Adapted from Figueroa et al, 2020 and Rossel et al., 2023

Figueroa, N.J., Figueroa, D.F. and Hicks, D., 2020. Phylogeography of Acartia tonsa Dana, 1849 (Calanoida: Copepoda) and phylogenetic reconstruction of the genus Acartia Dana, 1846. Marine Biodiversity, 50, pp.1-20.

Rossel, S., Kaiser, P., Bode‐Dalby, M., Renz, J., Laakmann, S., Auel, H., Hagen, W., Arbizu, P.M. and Peters, J., 2023. Proteomic fingerprinting enables quantitative biodiversity assessments of species and ontogenetic stages in Calanus congeners (Copepoda, Crustacea) from the Arctic Ocean. Molecular Ecology Resources, 23(2), pp.382-395.

DNA extraction: 
Samples were incubated in 30 μl chelex (InstaGene Matrix, Bio-Rad) for 50 min at 56°C, followed by a denaturation of the enzymes for 10 min at 96°C. Centrifuge samples @10,000rpm for 1min and carefully pipette out supernatant (avoid Chelex beads!). The resulting sample was used directly as DNA template for PCR amplification. 
Sample concentrations varied between 1-50ng/ul. 

Primers: COI barcode 
     F      LCO1409      GGTCAACAAATCATAAAGATATTGG      600bp        R      HCO2198      TAAACTTCAGGGTGACCAAAAAATCA      600bp   

PCR reaction mix:  
     Mastermix      1x        GoTaq 5x buffer      10ul        MgCl2 (25mM)      4ul        dNTPs (10uM each)      1ul        BSA (20mg/ml)      1ul        Primer (10uM)      2ul (each)        Mol. H2O      24.75ul        Taq      0.25ul        DNA template      5ul         Total      50ul    

Thermocycling conditions: 
94C, 5mins, 38x[94C, 45s; 42C, 45s; 72C, 1,20s], 72C, 7 min
SECTION: DNA extraction: General
1. DNA extraction: 
Unless otherwise stated, DNA extraction for marine metazoans is done with the Qiagen DNAeasy Mouse Blood and Tissue kit. 
https://www.qiagen.com/us/Resources/ResourceDetail?id=aa250d94-fc4b-4e27-bb74-d32391ff8a48&lang=en

The provided protocol is followed, apart from the final elution step: 
Pre-heated elution buffer (56C) is used to increase yield. 
An elution volume of 60ul is used to increase the final DNA concentration. 
The buffer is left to incubate on the membrane for 5 minutes. 

If sample yields are relatively high (>25ng/ul), gDNA samples are usually diluted by 1:10 to reduce contamination during PCR amplification.