License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 14, 2023
Last Modified: February 06, 2024
Protocol Integer ID: 90938
Keywords: jbaker@mbari.org
Funders Acknowledgement:
National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)
Grant ID: NASA grant NNX14AP62A
Abstract
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.
MIOP: Minimum Information about an Omics Protocol
MIOP: Minimum Information about an Omics Protocol
MIOP Term
Value
methodology category
omics analysis
project
Marine Biodiversity Observation Network (MBON)
purpose
PCR [OBI:0000415]
analyses
PCR [OBI:0000415]
geographic location
Monterey Bay [GAZ:00002509]
broad-scale environmental context
marine biome ENVO_00000447
local environmental context
oceanic epipelagic zone biome [ENVO:01000033]
environmental medium
sea water [ENVO:00002149] | DNA extraction [OBI:0000257]
This is a list of other protocols which should be known to users of this protocol. Please include the link to each related protocol.
ACRONYMS AND ABBREVIATIONS
ACRONYMS AND ABBREVIATIONS
ACRONYM / ABBREVIATION
DEFINITION
eDNA
environmental DNA
NTC
No Template Control
GLOSSARY
GLOSSARY
SPECIALISED TERM
DEFINITION
Content Cell
Content Cell
Content Cell
Content Cell
BACKGROUND
BACKGROUND
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 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.
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.
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).
Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure
Training requirements
Sterile technique, pipetting skills.
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.
EQUIPMENT
EQUIPMENT
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.
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.
PREPARATION
PREPARATION
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.
PCR
PCR
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.
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
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.
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
denaturation
98°C
30 seconds
1
denaturation
98°C
10 seconds
38 cycles
annealing
62°C
10 seconds
38 cycles
extension
72°C
30 seconds
38 cycles
final extension
72°C
5 minutes
1
HOLD
4°C
HOLD
1
Quality Control and Product Clean-up
Quality Control and Product Clean-up
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.
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.
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.
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.
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
3. Secondary 12S cycling parameters:
PCR step
Temperature
Duration
Repetition
denaturation
95°C
3 minutes
1
denaturation
95°C
15 seconds
15 cycles
annealing
60°C
30 seconds
15 cycles
extension
72°C
1 minute
15 cycles
final extension
72°C
3 minutes
1
HOLD
25°C
HOLD
1
QUALITY CONTROL
QUALITY CONTROL
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.
BASIC TROUBLESHOOTING GUIDE
BASIC TROUBLESHOOTING GUIDE
Identify known issues associated with the procedure, if any.
Provide troubleshooting guidelines when available.
REFERENCES
REFERENCES
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
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.