TITLE: Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)
AUTHORS: Kathleen Pitz, Jacoby Baker

[DESCRIPTION]

The 12S protocol is aimed at amplifying the hypervariable region of the mitochondrial DNA 12S rRNA gene in eukaryotes. The primers (MiFish-U-F & MiFish-U-R) used in this protocol were developed by Miya et al., 2015 for metabarcoding environmental DNA (eDNA) from fishes.

This protocol follows an updated version of the MiFish primer PCR protocol. The Platinum SuperFi II was designed to have high fideleity and have increased resistence to PCR inhibitors.




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SECTION: MIOP: Minimum Information about an Omics Protocol
1. 
  
MIOP Term
Value
   
methodology category

 
project

 
purpose

 
analyses

 
geographic location

 
broad-scale environmental context

 
local environmental context

 
environmental medium

 
target

 
creator

 
materials required

 
skills required

 
time required

 
personnel required

 
language

 
issued

 
audience

 
publisher

 
hasVersion

 
license

 
maturity level

  

SECTION: AUTHORS
2. 
  
PREPARED BY All authors known to have contributed to the preparation of this protocol, including those who filled in the template.
AFFILIATION
ORCID (visit https://orcid.org/ to register)
DATE
   
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SECTION: RELATED PROTOCOLS
3. 
  
PROTOCOL NAME AND LINK
VERSION The version of the protocol you linked to
RELEASE DATE This is the date corresponding to the version listed to the left
   
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SECTION: ACRONYMS AND ABBREVIATIONS
4. 
  
ACRONYM / ABBREVIATION
DEFINITION
   
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SECTION: GLOSSARY
5. 
  
SPECIALISED TERM
DEFINITION
   
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SECTION: BACKGROUND
6. Summary

The 12S protocol is aimed at amplifying the hypervariable region of the mitochondrial DNA 12S rRNA gene in eukaryotes. The primers (MiFish-U-F & MiFish-U-R) used in this protocol were developed by Miya et al., 2015 for metabarcoding environmental DNA (eDNA) from fishes.

This work was supported by the David and Lucile Packard Foundation, and NASA award 80NSSC21M0032 and NOAA award NA22NOS0120184 in support of the CeNCOOS MBON.
SECTION: BACKGROUND
7. Method description and rationale

This protocol follows an updated version of the MiFish primer PCR protocol. The Platinum SuperFi II was designed to have high fidelity and have increased resistance to PCR inhibitors.
SECTION: BACKGROUND
8. Spatial coverage and environment(s) of relevance

This protocol has been used to amplify extracted DNA from filtered sea water samples taken from marine coastal stations off the western coast of North America (primarily off of California).

sea water [ENVO:00002149]
http://purl.obolibrary.org/obo/ENVO_00002149
SECTION: BACKGROUND
9. Personnel Required

1 technician.
SECTION: BACKGROUND
10. Safety

Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure
SECTION: BACKGROUND
11. Training requirements

Sterile technique, pipetting skills.
SECTION: BACKGROUND
12. Time needed to execute the procedure

Total time is 7 hours.
PCR preparation and running the PCR protocol takes 3 hours. Running the following gel is 1 hour, bead cleanup setup preparation and process takes 2 hours, and then another gel is run for 1 hour.
SECTION: EQUIPMENT
13. 
  
DESCRIPTION e.g. filter
PRODUCT NAME AND MODEL Provide the official name of the product
MANUFACTURER Provide the name of the manufacturer of the product.
QUANTITY Provide quantities necessary for one application of the standard operating procedure (e.g. number of filters).
REMARK For example, some of the consumable may need to be sterilized, some commercial solution may need to be diluted or shielded from light during the operating procedure.
   
Durable equipment




 
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Consumable equipment




 
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Chemicals




 
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SECTION: STANDARD OPERATING PROCEDURE
14. In the following SOP, please use the exact names of equipment as noted in the table above.

Provide a step-by-step description of the protocol. The identification of difficult steps in the protocol and the provision of recommendations for the execution of those steps are encouraged.
SECTION: PREPARATION
15. BEFORE STARTING

Disinfect work surfaces with 10% bleach or RNase Away followed by a MilliQ / DI water rinse and 70% ethanol wipe. Clean pipet surfaces with RNase Away and ethanol wipe.
UV pipets, molecular grade water, and tube racks for 30 minutes prior to starting protocol.

SECTION: PCR
16. 1. eDNA template & PCR processing were performed at the Monterey Bay Aquarium Research Institute (MBARI). PCR reactions for the 12S locus were performed with a two-step amplification protocol for each sample using the MiFish_U primers (Miya et al. 2015) with Fluidigm adapters CS1 & CS2. All primers listed in the 5’ to 3’ direction. MiFish primers are in bold.

Fluidigm CS1+MiFish_U (forward):
ACACTGACGACATGGTTCTACA GTCGGTAAAACTCGTGCCAGC
Fluidigm CS2+Mifish_U (reverse):
TACGGTAGCAGAGACTTGGTCT CATAGTGGGGTATCTAATCCCAGTTTG

  
PCR Primer Name
Direction
Sequence (5’ -> 3’)
   
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forward
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reverse
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SECTION: Quality Control and Product Clean-up
20. 1. After primary PCR amplification of the marker region the PCR product was run through a 2% agarose gel to confirm the presence of target bands (~270 bp) and absence of non-specific amplification (bacterial band ~370 bp) across environmental samples.

Primary PCR clean-up (Bead Clean-up Protocol)
SECTION: QUALITY CONTROL
26. An agarose gel was run after secondary PCR to confirm the presence of target bands and absence of non-specific amplification across environmental samples as well as the absence of amplification in NTCs.

Products from the protocol are then used to create a pooled library and sequenced following a separate sequencing protocol (such as "Environmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS Protocol with size selection V.2", dx.doi.org/10.17504/protocols.io.eq2lyj1kelx9/v2 )
SECTION: BASIC TROUBLESHOOTING GUIDE
27. Identify known issues associated with the procedure, if any.

Provide troubleshooting guidelines when available.
SECTION: REFERENCES
28. Miya M et al. 2015 MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. R.Soc.opensci. 2: 150088. http://dx.doi.org/10.1098/rsos.150088
Kawato, M., Yoshida, T., Miya, M., Tsuchida, S., Nagano, Y., Nomura, M., Yabuki, A., Fujiwara, Y. and Fujikura, K., 2021. Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish. MethodsX, 8, p.101238. https://doi.org/10.1016/j.mex.2021.101238
Platinum SuperFi II PCR Master Mix User Guide
SECTION: APPENDIX A: DATASHEETS
29. Link templates (e.g. preformatted spreadsheets) used to record measurements and report on the quality of the data as well as any documents such as manufacturer specifications, images, etc that support this protocol. Please include a short note describing the document’s relevance.
SECTION: PCR
19. 4. Primary 12S cycling parameters, using the manufacturer’s recommendation with a slight modification. The Platinum SuperFi II is designed to be used with a universal 60°C annealing temperature, however, we found we had better results with an annealing temperature of 62°C to reduce bacterial co-amplification:

  
PCR step
Temperature
Duration
Repetition
   
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SECTION: PCR
17. 2. The primary PCR amplifications were carried out in singleton 50 μl reactions using:
25 μL 2X Platinum SuperFi II PCR MM
3 μL forward primer (10 μM)
3 μL reverse primer (10 μM)
3 μL eDNA extract template
16 μL molecular-grade, nuclease-free water
SECTION: PCR
18. 3. PCR reactions were performed in 96-well plates with a no-template control (NTC) for each PCR plate, for a total of 3 PCR negative controls. An artificial community was used as a positive control.
SECTION: Quality Control and Product Clean-up
21. 2. Primary PCR products were purified and size selected using the Agencourt AMPure XP bead system (Beckman Coulter, USA) at 0.9x volume beads to product.
SECTION: Quality Control and Product Clean-up
22. 3. A second agarose gel was run to confirm primer removal and retention of target amplicons after purification. NTCs were also tested using a Qubit dsDNA 1x high sensitivity kit to ensure no amplification.

SECTION: Secondary Amplification
23. The following steps are performed by MSU’s RTSF Genomics Core

1. Secondary amplification and NGS were performed at Michigan State University’s Research Technology Support Facility (RTSF). An aliquot of 20 μL from each purified primary PCR product was sent to RTSF Genomics Core at MSU for secondary PCR amplification with primers which targeted the CS1/CS2 ends of the primary PCR products and added dual indexed, Illumina compatible adapters with barcodes.

PE1-BC-CS1 (forward): 
AATGATACGGCGACCACCGAGATCT-[i5-BC(index 2)]-ACACTGACGACATGGTTCTACA
PE2-BC-CS2 (reverse): 
CAAGCAGAAGACGGCATACGAGAT-[i7-BC(index 1)]-TACGGTAGCAGAGACTTGGTCT

  
PCR Primer Name
Direction
Sequence (5’ -> 3’)
   
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forward
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reverse
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SECTION: Secondary Amplification
24. 2. The secondary PCR amplifications were carried out in 15 μL reactions, using 1 μL of primary PCR product.
6 μl 2.5X HotMaster Mix
7 μl DI water
1 μl Primer Mix (6uM)
1 μl primary eDNA PCR product
SECTION: Secondary Amplification
25. 3. Secondary 12S cycling parameters:

  
PCR step
Temperature
Duration
Repetition
   
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