TITLE: Environmental DNA (eDNA) COI Metabarcoding PCR Protocol
AUTHORS: Jacoby Baker, Kathleen Pitz

[DESCRIPTION]
This protocol is aimed at amplifying the cytochrome c oxidase subunit I (COI) mitochondrial gene in eukaryotes. The primers (forward: mlCOIintF, reverse: HCO2198) utilized in this protocol are based on the primers utilized in Leray et al. 2013 (forward) and Folmer et al. 1994 (reverse).

Primers used: Fluidigm CS1+mlCOIinfF, Fluidigm CS2+HCO2198
Amplicons generated using this protocol can then be sequenced using the Illumina platform.

Primary PCR amplicon products are then sent to Michigan State University's (MSU) Research Technology Support Facility (RTSF) for indexing, pooling, and sequencing.

This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office.

[STEPS]
SECTION: Minimum Information about an Omics Protocol (MIOP)
1. 
  
MIOP Term
Value
   
methodology category

 
project

 
purpose

 
analyses

 
geographic location

 
broad-scale environmental context

 
local environmental context

 
environmental medium

 
target
18S gene
 
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
   
Jacoby Baker
MBARI

yyyy-mm-dd
 
Kobun Truelove
MBARI

yyyy-mm-dd
 
Kathleen Johnson Pitz
MBARI
0000-0002-4931-8592
2022-04-25
  

SECTION: PROTOCOL REVISION RECORD
3. Version numbers start at “1.0.0” when the protocol is first completed and will increase when changes that impact the outcome of the procedure are made (patches: 1.0.1; minor changes: 1.1.0; major changes: 2.0.0). Please store all versions in the gDrive folder designated to your institute.

  
VERSION
RELEASE DATE This is the date when a given protocol version was finalised
DESCRIPTION OF REVISIONS Please include a brief description of what was changed relative to the previous version
   
1.0.0
2022-04-25
Initial release
    

SECTION: RELATED EXTERNAL PROTOCOLS
4. This is a list of other protocols that are not in your folder which should be known to users of this protocol. These include, e.g., kit manuals. Please upload all relevant external protocols to Appendix A and link to them here.

  
EXTERNAL PROTOCOL NAME AND LINK
ISSUER / AUTHOR Please note who authored the protocol (this may also be a company name)
ACCESS DATE This is the date you downloaded or scanned the protocol and uploaded it.
   
Environmental DNA (eDNA) 18S metabarcoding Illumina MiSeq NGS PCR Protocol V.2 https://dx.doi.org/10.17504/protocols.io.n2vdge6 dx.doi.org/10.17504/protocols.io.n2vdge6
Collin Closek, Anni Djurhuus, Katie Pitz, Ryan Kelly, Reiko Michisaki, Kristine Walz, Hilary Starks, Francisco Chavez, Alexandria Boehm, Mya Breitbart
yyyy-mm-dd
 
18S Illumina Amplicon Protocol https://earthmicrobiome.org/protocols-and-standards/18s/ dx.doi.org/10.17504/protocols.io.nuvdew6
Earth Microbiome Project
yyyy-mm-dd
  

SECTION: ACRONYMS AND ABBREVIATIONS
5. 
  
ACRONYM / ABBREVIATION
DEFINITION
   
MBARI
Monterey Bay Aquarium Research Institute
 
PCR
polymerase chain reaction
 
NTC
no template control
  

SECTION: GLOSSARY
6. 
  
SPECIALISED TERM
DEFINITION
   
amplicon
A piece of DNA or RNA that is the source and/or product of amplification or replication events (https://en.wikipedia.org/wiki/Amplicon)
  

SECTION: BACKGROUND
7. This protocol is aimed at amplifying the cytochrome c oxidase subunit I (COI) mitochondrial gene in eukaryotes. 


This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office.
SECTION: Summary
8. This protocol is aimed at amplifying the cytochrome c oxidase subunit I (COI) mitochondrial gene in eukaryotes. The primers (forward: mlCOIintF, reverse: HCO2198) utilized in this protocol are based on the primers utilized in Leray et al. 2013 (forward) and Folmer et al. 1994 (reverse).

PCR reactions for COI were run with Fluidigm two-step amplification protocol for each sample
SECTION: Method description and rationale
9. This method is applied because of its ability to amplify the target region (COI) across many different groups of organisms, the target region’s ability to discriminate between different taxa, and the common research application of this primer set allowing the data to be compared to a reference database and other published environmental datasets.
SECTION: Spatial coverage and environment(s) of relevance
10. ocean [ENVO:00000015]
freshwater lake [ENVO:00000021]
SECTION: PERSONNEL REQUIRED
11. 1 technician
SECTION: EQUIPMENT
12. 
  
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




 
ultraviolet light source [OBI:0002900]




 
PCR instrument [OBI:0000989]




 
electrophoresis system [OBI:0001053]




 
fluorometer [OBI:0400143]
FMAX Fluorometer
Molecular Devices

with SoftMaxPro v1.3.1
 
Consumable equipment




 
Agarose gel


2

 
Agencourt AMPure XP bead system

Beckman Coulter, USA


 
Quant-It Picogreen dsDNA Assay

Life Technologies


 
Chemicals




 
10% Bleach




 
70% Ethanol




 
RNase Away




 
Amplitaq Gold Fast PCR mastermix




 
molecular-biology grade water




 
forward and reverse primers (5 μM)




  

SECTION: Preparation
13. 
Disinfect work surfaces with 10% bleach, followed by 70% ethanol.
RNase Away and pipets with RNase Away
UV pipets, molecular grade water, and tube racks for 20 minutes prior to starting protocol.

SECTION: PCR
14. PCR reactions were run in single 75ul reactions for each sample using the 26bp primers (forward: mlCOIintF, reverse: HCO2198) utilized in this protocol are based on the primers utilized in Leray et al. 2013 (forward) and Folmer et al. 1994 (reverse) with Fluidigm adapters CS1 & CS2. All primers listed in the 5’ to 3’ direction.

  
PCR Primer Name
Direction
Sequence (5’ -> 3’)
   
Euk1391F and Fluidigm CS1
forward
ACACTGACGACATGGTTCTACAGTACACACCGCCCGTC
 
EukBr and Fluidigm CS2
reverse
TACGGAGCAGAGACTTGGTCTTGATCCTTCTGCAGGTTCACCTAC
  

PCR reactions were run in 96-well plates with a NTC run in singleton for each plate

COI thermal cycling parameters (note: this is a touchdown PCR protocol)
These parameters use a normal ramp speed

  
PCR step
Temperature
Duration
Repetition
   
denature
95° C
10 minutes
1
 
denature
94° C
45 seconds
35
 
anneal
57° C
30 seconds
35
 
extension
68 °C
90 seconds
35
 
extension
72° C
10 minutes
1
 
hold
4° C
infinity
1
  

SECTION: Quality control, PCR clean-up
16. After PCR amplification of the marker region, PCR products were run through an agarose gel to confirm the presence of target bands and absense of non-specific amplification across environmental samples as well as the absence of amplification in no-template controls (NTCs).

PCR products were purified and size selected using the Agencourt AMPure XP bead system (Beckman Coulter, USA).

A second agarose gel was run to confirm primer removal and retention of target amplicons after purification.

Purified products were then quantified using Quant-It Picogreen dsDNA Assay (Life Technologies) on an fmax Molecular Devices Fluorometer with SoftMaxPro v1.3.1

SECTION: REFERENCES
18. Leray M, Yang JY, Meyer CP, Mills SC, Agudelo N, Ranwez V, Boehm JT, Machida RJ. (2013) A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents. Frontiers in zoology, 10(1),1-4.
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299.
SECTION: APPENDIX A: DATASHEETS
19. 
SECTION: Next Steps
17. From here, the amplicon products will move onto the indexing PCR step, normalization, pooling, and sequencing.
SECTION: PCR
15. Reaction Mixture: PCR reagents, volumes, initial and final concentrations
Total volume per reaction 75 μl