TITLE: 18S V9 PCR
AUTHORS: Kathleen Pitz

[DESCRIPTION]
This protocol is aimed at amplifying the 18S rRNA hypervariable region 9 (18S V9) in eukaryotes with a focus on microbial eukaryotes. Amplicons generated using this protocol can then be sequenced using the Illumina platform. The primers (1391F, EukBr) utilized in this protocol are based on the primer utilized in Amaral-Zettler et al. (2009), Stoek et al. (2010), and the Earth Microbiome Project (EMP).

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 PROTOCOLS IN YOUR FOLDER
4. This is a list of other protocols deposited in your folder which should be known to users of this protocol. For example, if you create a derivative or altered protocol, you would link to the original protocol in the section below. Please include the link to each related protocol. Also include the version number of that protocol when you linked to it.

  
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
   
protocol_18S_secondary_amplification.md

yyyy-mm-dd
 
protocol_18S_sequencing.md

yyyy-mm-dd
  

SECTION: RELATED EXTERNAL PROTOCOLS
5. 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
6. 
  
ACRONYM / ABBREVIATION
DEFINITION
   
MBARI
Monterey Bay Aquarium Research Institute
 
PCR
polymerase chain reaction
 
NTC
no template control
  

SECTION: GLOSSARY
7. 
  
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
8. This protocol is aimed at amplifying the 18S rRNA hypervariable region 9 (18S V9) in eukaryotes with a focus on microbial eukaryotes. Amplicons generated using this protocol can then be sequenced using the Illumina platform. The primers (1391F, EukBr) utilized in this protocol are based on the primer utilized in Amaral-Zettler et al. (2009), Stoek et al. (2010), and the Earth Microbiome Project (EMP).


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
9. This method uses PCR to amplify the 18S V9 region using primers 1391F and EukBr from Amaral-Zettler et al 2009 and the Earth Microbiome Project (EMP).
SECTION: Method description and rationale
10. This method is applied because of its ability to amplify the target region (18S V9) 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
11. ocean [ENVO:00000015]
freshwater lake [ENVO:00000021]
SECTION: PERSONNEL REQUIRED
12. 1 technician
SECTION: Safety
13. 
 Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure
 
SECTION: Training requirements
14. 
 Specify technical training required for the good execution of the procedure.
 
SECTION: Time needed to execute the procedure
15. 

 Specify how much time is necessary to execute the procedure.
 
SECTION: EQUIPMENT
16. 
  
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
17. 
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
18. PCR reactions were run in single 75ul reactions for each sample using 12-basepair Golay barcoded reverse primers [Amaral-Zettler et al. (2009), Stoek et al. (2010), Earth Microbiome Project] with Fluidigm adapters CS1 & CS2. All primers listed in the 5’ to 3’ direction.

3 μl DNA extract template
37.5 μl Amplitaq Gold Fast PCR mastermix (Applied Biosystems)
3 μl each of forward and reverse primers (5 μM)
28.5 μl molecular-biology grade water

  
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

18S thermal cycling parameters
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
19. 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: Basic troubleshooting guide
20. 

 Identify known issues associated with the procedure, if any.

Provide troubleshooting guidelines when available.
 
SECTION: REFERENCES
21. Amaral-Zettler LA, McCliment EA, Ducklow HW, Huse SM (2009) A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes. PLOS ONE 4(7): e6372. https://doi.org/10.1371/journal.pone.0006372


Stoeck, T., Bass, D., Nebel, M., Christen, R., Jones, M. D. M., Breiner, H.-W., & Richards, T. A. (2010). Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Molecular Ecology, 19 Suppl 1, 21–31. https://doi.org/10.1111/j.1365-294X.2009.04480.x


Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S. M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J. A., Smith, G., & Knight, R. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621–1624. http://doi.org/10.1038/ismej.2012.8
SECTION: APPENDIX A: DATASHEETS
22.