Sep 04, 2024
Environmental DNA (eDNA) indexing PCR protocol at MSU
- Jacoby Baker1,
- Kathleen Pitz1
- 1MBARI
- Better Biomolecular Ocean Practices (BeBOP)
Protocol Citation: Jacoby Baker, Kathleen Pitz 2024. Environmental DNA (eDNA) indexing PCR protocol at MSU. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvme1z5g3p/v1
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: September 03, 2024
Last Modified: September 04, 2024
Protocol Integer ID: 106884
Keywords: [email protected], indexing pcr protocol, indexing pcr, primary pcr amplification protocol, secondary pcr primer, genomics core for indexing, primary pcr product, environmental dna, amplified dna products of each sample, pcr, pcr product, amplified dna product, sequencing result, sequencing protocol, genomics core, mitochondrial gene, sequencing, dna, indexing
Funders Acknowledgements:
National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)
Grant ID: NASA grant NNX14AP62A
Abstract
This sequencing protocol is intended to directly follow and use the PCR products of a primary PCR amplification protocol (e.g., 12S rRNA, 18S rRNA V9, cytochrome c oxidase subunit I (COI) mitochondrial gene). Primary PCR products were produced at MBARI and sent to Michigan State University's (MSU) Research Technology Support Facility (RTSF) Genomics Core for indexing, pooling, and sequencing.
Secondary PCR primers used: PE1-BC-CS1, PE2-BC-CS2
This indexing PCR protocol is used to uniquely index the amplified DNA products of each sample. This is necessary so the sequencing results can be demultiplexed and the reads can be properly identified and assigned to each individual sample.
Troubleshooting
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] | PCR product [OBI:0000406] | |
| target | PCR product [OBI:0000406] | |
| creator | Jacoby Baker, https://orcid.org/0000-0002-0673-7535 | |
| materials required | agarose gel electrophoresis system [OBI:0001134] | PCR instrument [OBI:0000989] | |
| skills required | sterile technique | pipetting skills | |
| time required | 360 | |
| personnel required | 1 | |
| language | en | |
| issued | 2024-09-04 | |
| audience | scientists | |
| publisher | Monterey Bay Aquarium Research Institute, Chavez Lab | |
| hasVersion | V.1 | |
| license | CC BY 4.0 | |
| maturity level | Mature |
See https://github.com/BeBOP-OBON/miop/blob/main/model/schema/terms.yaml for list and definitions.
AUTHORS
| 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 | 0000-0002-0673-7535 | 2023-11-07 | |
| N. Kobun Truelove | MBARI | 0000-0002-2236-1849 | 2023-11-07 | |
| Kathleen J. Pitz | MBARI | 0000-0002-4931-8592 | 2023-11-07 | |
| Francisco Chavez | MBARI | 2023-11-07 |
MBARI : Monterey Bay Aquarium Research Institute, Moss Landing, CA
RELATED PROTOCOLS
Example protocols for primary PCR, secondary PCR, and bead cleanups:
| PROTOCOL NAME AND LINK | ISSUER / AUTHOR | RELEASE DATE This is the date corresponding to the version listed to the left | |
| https://mbari-bog.github.io/MBON-Protocols/eDNA_COI_PCR_V2.html | Jacoby Baker | 2023-11-07 | |
| https://mbari-bog.github.io/MBON-Protocols/Bead_cleanup.html | Jacoby Baker | 2023-11-07 |
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
| ACRONYM / ABBREVIATION | DEFINITION | |
| eDNA | environmental DNA | |
| NTC | No Template Control | |
| PCR | polymerase chain reaction |
GLOSSARY
| 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) |
BACKGROUND
Summary
The PCR protocol is used to uniquely index PCR amplicon products from the primary taxa-targeting PCR step using gene-targeting PCR primers (e.g., 12S rRNA, 18S rRNA V9, cytochrome c oxidase subunit I (COI) mitochondrial gene).
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 is performed at Michigan State University's (MSU) Research Technology Support Facility(RTSF) Genomics Core (https://rtsf.natsci.msu.edu/genomics/) to prepare amplicon projects for MiSeq sequencing.
Spatial coverage and environment(s) of relevance
This protocol has been used to uniquely index amplified 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]
Personnel Required
1 technician.
Safety
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 6 hours.
PCR preparation and running the PCR protocol takes 1 hours. Running the following gel is 1 hour, bead cleanup setup preparation and process takes 2 hours, and then sample normalization and pooling takes 2 hours.
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. | F | |
| Durable equipment | ||||||
| Agarose gel electrophoresis system | ||||||
| PCR Thermal Cycler | ||||||
| Consumable equipment | ||||||
| PCR plates | SuperPlate PCR Plate, 96-well, semi-skirted | Thermofisher Scientific | https://www.thermofisher.com/order/catalog/product/AB2400#/AB2400 | |||
| Plate seals | PCR Plate Seals | Bio Rad | https://www.bio-rad.com/en-us/product/pcr-plate-seals?ID=OC0OOTMNI | |||
| Chemicals | ||||||
| OneTaq® Hot Start 2X Master Mix with Standard Buffer | OneTaq® Hot Start 2X Master Mix with Standard Buffer | NEB | https://www.neb.com/en-us/products/m0484-onetaq-hot-start-2x-master-mix-with-standard-buffer | |||
| molecular-biology grade water | ||||||
| Illumina compatible dual indexed adapters with barcodes | ||||||
| Amplicon product from the primary PCR reaction |
STANDARD 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
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.
Primary PCR
eDNA template & PCR processing were performed at the Monterey Bay Aquarium Research Institute (MBARI). PCR reactions were performed with a two-step amplification protocol for each sample using the gene-targeting primers (e.g., 12S MiFish, 18S V9, COI, or 16S V4-V5) with Fluidigm adapters CS1 & CS2. The resulting amplicon product from the primary PCR was bead cleaned, quantified, then sent to MSU for indexing. Below are the steps for the secondary amplificaiton. Library normaliztion, pooling, and sequencing are described in a complementary protocol.
Secondary 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.
- 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’) | |
| PE1-BC-CS1 | forward | AATGATACGGCGACCACCGAGATCT-[i5-BC(index 2)]-ACACTGACGACATGGTTCTACA | |
| PE2-BC-CS2 | reverse | CAAGCAGAAGACGGCATACGAGAT-[i7-BC(index 1)]-TACGGTAGCAGAGACTTGGTCT |
Secondary Indexing Thermal Cycling Parameters:
The secondary/indexing PCR amplifications were carried out in 15 μL reactions, using 1 μL of primary PCR product.
| 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 |
Reaction Mixture: PCR reagents, volumes, initial and final concentrations
Total volume per reaction 15 μl
| Reagent | Volume | Initial Concentration | Final Concentration | |
| OneTaq® Hot Start 2X Master Mix with Standard Buffer | 6 μl | 2X | 1X | |
| Primer Mix (6uM) | 1 μl | 6 μM | 0.4 μM | |
| molecular-biology grade water | 7 μl | - | - | |
| primary eDNA PCR product | 1 μl | variable | < 1,000 ng |
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.
REFERENCES
- Kozich, J. J. et al. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and environmental microbiology 79, 5112–5120 (2013).
APPENDIX A: DATASHEETS
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.