TITLE: Environmental DNA (eDNA) indexing PCR  protocol at MSU
AUTHORS: Jacoby Baker, Kathleen Pitz

[DESCRIPTION]
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.


[STEPS]
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. Example protocols for primary PCR, secondary PCR, and bead cleanups:
  
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 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.
SECTION: BACKGROUND
7. 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.
SECTION: BACKGROUND
8. 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]
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 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.
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: Primary PCR
16. 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.
SECTION: QUALITY CONTROL
20. 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.
SECTION: REFERENCES
21. 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).
https://www.neb.com/en-us/protocols/2012/09/10/onetaq-hot-start-2x-master-mix-with-standard-buffer-m0484
SECTION: APPENDIX A: DATASHEETS
22. 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: Secondary Amplification
17. 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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reverse
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SECTION: Secondary Amplification
18. 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
   
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SECTION: Secondary Amplification
19. Reaction Mixture: PCR reagents, volumes, initial and final concentrations
Total volume per reaction 15 μl