Environmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS Protocol with size selection

Kathleen Pitz; Jacoby Baker

Dec 21, 2023

Version 2

Environmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS Protocol with size selection V.2

Version 1 is forked from SEQUENCING Protocol Template

DOI

dx.doi.org/10.17504/protocols.io.eq2lyj1kelx9/v2

Kathleen Pitz¹,
Jacoby Baker¹

¹MBARI

Kathleen Pitz

Monterey Bay Aquarium Research Institute

DOI: dx.doi.org/10.17504/protocols.io.eq2lyj1kelx9/v2

Protocol Citation: Kathleen Pitz, Jacoby Baker 2023. Environmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS Protocol with size selection. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyj1kelx9/v2Version created by Kathleen Pitz

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: December 18, 2023

Last Modified: February 06, 2024

Protocol Integer ID: 92455

Funders Acknowledgement:

David and Lucile Packard Foundation

NASA

Grant ID: 80NSSC21M0032

NOAA

Grant ID: NA22NOS0120184

Abstract

This sequencing protocol is intended to directly follow and use the PCR products of the protocol:
"Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)" which amplifies the hypervariable region of the mitochondrial DNA 12S rRNA gene in eukaryotes. 

This protocol creates a pooled library which is then size selected using a Blue Pippin or Pippin HT to select for the vertebrate/fish band (~350 bp) and remove the co-amplified bacterial band (~435 bp).  Then the pooled product is sequenced on an Illumina MiSeq v2 in a 2x250bp paired end format.

MIOP: Minimum Information about an Omics Protocol

MIOP TermValue
methodology categoryomics analysis
projectMarine Biodiversity Observation Network (MBON)
purposetaxonomic diversity assessment by targeted gene survey [OBI:0001960]
analysesDNA sequencing assay [OBI:0000626]
geographic locationMonterey Bay [GAZ:00002509]
broad-scale environmental contextmarine biome ENVO_00000447
local environmental contextoceanic epipelagic zone biome [ENVO:01000033]
environmental mediumPCR product [OBI:0000406]
targetActinopterygii [NCBITaxon:7898]
creatorJacoby Baker, https://orcid.org/0000-0002-0673-7535
materials requiredIllumina MiSeq | Blue Pippin
skills required
time required
personnel required1
languageen
issued2023-11-14
audiencescientists
publisherMonterey Bay Aquarium Research Institute, Chavez Lab
hasVersionV.3
licenseCC BY 4.0
maturity levelMature
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.AFFILIATIONORCID (visit https://orcid.org/ to register)DATE
Jacoby BakerMBARI0000-0002-0673-75352023-11-07
N. Kobun TrueloveMBARI0000-0002-2236-18492023-11-07
Kathleen J. PitzMBARI0000-0002-4931-85922023-11-07
Francisco ChavezMBARI2023-11-07

RELATED PROTOCOLS

PROTOCOL NAME AND LINKISSUER / AUTHORRELEASE / ACCESS DATE
https://mbari-bog.github.io/MBON-Protocols/eDNA_12S_SupFi2_PCR_V3.htmlJacoby Baker2023-11-07
Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)Jacoby Baker2023-11-07
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 / ABBREVIATIONDEFINITION
eDNAenvironmental DNA

GLOSSARY

SPECIALISED TERMDEFINITION

BACKGROUND

Summary

This sequencing protocol is intended to directly follow and use the PCR products of the protocol:
"Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)" which amplifies the hypervariable region of the mitochondrial DNA 12S rRNA gene in eukaryotes. 

The primers (MiFish-U-F & MiFish-U-R) used in the PCR protocol were developed by Miya et al., 2015 for metabarcoding environmental DNA (eDNA) from fishes.

This work was supported by the David and Lucile Packard Foundation, and NASA award 80NSSC21M0032 and NOAA award NA22NOS0120184 in support of the CeNCOOS MBON.

Method description and rationale

This protocol creates a pooled library which is then size selected using a Blue Pippin or Pippin HT to select for the vertebrate/fish band (~350 bp) and remove the co-amplified bacterial band (~435 bp).  Then the pooled product is sequenced on an Illumina MiSeq v2 in a 2x250bp paired end format.

Spatial coverage and environment(s) of relevance

This protocol has been used to sequence 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

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

Time needed to execute the procedure

EQUIPMENT

DESCRIPTION e.g. filterPRODUCT NAME AND MODEL Provide the official name of the productMANUFACTURER 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
Illumina MiSeqIllumina MiSeqIllumina
TapeStationAgilent 4200 TapeStation HS DNA1000Agilent
Blue PippinBlue PippinSageScience
Consumable equipment
Invitrogen SequalPrep Normalization PlateInvitrogen SequalPrep Normalization PlateThermoFisher Scientific
Chemicals
Library Quantification KitInvitrogen Collibri Library Quantification qPCR assaysInvitrogen
Pippin HT kitHTC2010Sage Science

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

Follow steps in the protocol "Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)" through secondary amplification and QC of 12S PCR product.

Pool Library

1. After secondary PCR, products were run through Invitrogen SequalPrep Normalization Plate (ThermoFisher Scientific) using manufacturer’s protocol to create pooled library.

2. The library pools were QC’d and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000 and Invitrogen Collibri Library Quantification qPCR assays.

Size selection of final library

1. After the pooled library was QC’d, the library was size selected with either a Blue Pippin or Pippin HT to select for the vertebrate/fish band (~350 bp) and remove the co-amplified bacterial band (~435 bp).

2. After size selection, the pooled library was QC’d again to confirm selection of the correct band and new amplicon concentrations.

SEQUENCING

The pooled product for the genetic locus was loaded on a standard MiSeq v2 flow cell and sequenced in a 2x250bp paired end format using a v2 500-cycle MiSeq reagent cartridge.

2. The MiSeq run was performed with a 20% PhiX spike added.

3. Primers complementary to the Fluidigm CS1 & CS2 oligomers were added to appropriate wells of the reagent cartridge to server as sequencing and index read primers.

12S Sequencing primers (5’ to 3’ direction):
FL1-CS1(read1) 
A+CA+CTG+ACGACATGGTTCTACA

FL1-CS2(read2) 
T+AC+GGT+AGCAGAGACTTGGTCT

FL2-CS1rc 
T+GT+AG+AACCATGTCGTCAGTGT

FL2-CS2rc(index) 
A+GAC+CA+AGTCTCTGCTACCGTA

 
Sequencing Primer NameDirectionSequence (5’ -> 3’)
FL1-CS1read1A+CA+CTG+ACGACATGGTTCTACA
FL1-CS2read2T+AC+GGT+AGCAGAGACTTGGTCT
FL2-CS1rcT+GT+AG+AACCATGTCGTCAGTGT
FL2-CS2rcindexA+GAC+CA+AGTCTCTGCTACCGTA

 
 

4. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0

QUALITY CONTROL

None

BASIC TROUBLESHOOTING GUIDE

None

REFERENCES

None

APPENDIX A: DATASHEETS

None

	MIOP Term	Value
	methodology category	omics analysis
	project	Marine Biodiversity Observation Network (MBON)
	purpose	taxonomic diversity assessment by targeted gene survey [OBI:0001960]
	analyses	DNA sequencing assay [OBI:0000626]
	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	PCR product [OBI:0000406]
	target	Actinopterygii [NCBITaxon:7898]
	creator	Jacoby Baker, https://orcid.org/0000-0002-0673-7535
	materials required	Illumina MiSeq \| Blue Pippin
	skills required
	time required
	personnel required	1
	language	en
	issued	2023-11-14
	audience	scientists
	publisher	Monterey Bay Aquarium Research Institute, Chavez Lab
	hasVersion	V.3
	license	CC BY 4.0
	maturity level	Mature

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

PROTOCOL NAME AND LINK	ISSUER / AUTHOR	RELEASE / ACCESS DATE
https://mbari-bog.github.io/MBON-Protocols/eDNA_12S_SupFi2_PCR_V3.html	Jacoby Baker	2023-11-07
Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)	Jacoby Baker	2023-11-07

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
Illumina MiSeq	Illumina MiSeq	Illumina
TapeStation	Agilent 4200 TapeStation HS DNA1000	Agilent
Blue Pippin	Blue Pippin	SageScience
Consumable equipment
Invitrogen SequalPrep Normalization Plate	Invitrogen SequalPrep Normalization Plate	ThermoFisher Scientific
Chemicals
Library Quantification Kit	Invitrogen Collibri Library Quantification qPCR assays	Invitrogen
Pippin HT kit	HTC2010	Sage Science

Sequencing Primer Name	Direction	Sequence (5’ -> 3’)
FL1-CS1	read1	A+CA+CTG+ACGACATGGTTCTACA
FL1-CS2	read2	T+AC+GGT+AGCAGAGACTTGGTCT
FL2-CS1rc		T+GT+AG+AACCATGTCGTCAGTGT
FL2-CS2rc	index	A+GAC+CA+AGTCTCTGCTACCGTA

	ACRONYM / ABBREVIATION	DEFINITION
	eDNA	environmental DNA

	SPECIALISED TERM	DEFINITION

Public workspaceEnvironmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS Protocol with size selection V.2

Environmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS Protocol with size selection V.2