A	B
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	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, http://orcid.org/0000-0002-0673-7535
materials required	Illumina MiSeq
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

See http://github.com/BeBOP-OBON/miop/blob/main/model/schema/terms.yaml for list and definitions.

A	B	C	D
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

A	B	C
http://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

This is a list of other protocols which should be known to users of this protocol. Please include the link to each related protocol.

A	B
eDNA	environmental DNA

A	B

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

A	B	C	D	E
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

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.

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.

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.

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.

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)

• FL1-CS2(read2)

• FL2-CS1rc

• FL2-CS2rc(index)

A	B	C
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

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

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