Dataset: Quantitative PCR: Field Validation

Name: Measurements of mixed field samples used for qPCR estimates and estimating biomass of DNA copy number in bulk samples
Published: 2022-02-03
Keywords: oceans

Cite This Dataset

DOI:10.26008/1912/bco-dmo.869225.1

Spatial Extent: N:21.45 E:-157.8 S:21.45 W:-157.8

Temporal Extent: 2015-05 - 2015-07

Project:

EAGER: New molecular methods for studying copepod nauplii in the field (EAGER: Copepod nauplii)

Principal Investigator:

Erica Goetze (University of Hawaiʻi at Mānoa, SOEST)

Co-Principal Investigator:

Petra H. Lenz (University of Hawaiʻi at Mānoa, PBRC)

Karen E. Selph (University of Hawaiʻi at Mānoa, SOEST)

Contact:

Michelle Jungbluth

BCO-DMO Data Manager:

Shannon Rauch (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2022-02-03

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Measurements of mixed field samples used for qPCR estimates and estimating biomass of DNA copy number in bulk samples

Abstract:

Quantitative PCR data from developing four assays to quantify copepod naupliar biomass in Kaneohe Bay, Hawaii. These data are from measurements of mixed field samples that were used for qPCR estimates and estimating biomass of DNA copy number in bulk samples.

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Archival Copy

Version Date	Archive	Persistent Identifier (PID)	Date PID Issued
2022-02-03	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.26008/1912/bco-dmo.869225.1	2022-02-09

Methods & Sampling

Methodology:
Complete methodology is detailed in Jungbluth et al. (accepted 2022, Limnology and Oceanography Methods). Briefly, qPCR assays were developed for four copepod species: Parvocalanus crassirostris, Bestiolina similis, Oithona simplex, and Oithona attenuata. The assays were optimized and applied to mixed field samples to estimate biomass of nauplii of each species, over 5 orders of magnitude in length-based biomass and DNA copy number measured with qPCR.

Sampling and Analytical Procedures:
Zooplankton samples were either collected from the field with a Niskin bottle or 150 µm mesh plankton net with filtering cod end, or cultured in the laboratory, then preserved in 95% molecular ethyl alcohol and stored in the freezer until processing. Field samples were collected from southern Kāne‘ohe Bay on the island of Oahu, Hawai‘i. DNA was extracted from individual copepods of all focal and non-focal species from the area and used to assess whether our qPCR assays were working as expected. Dilutions of the various focal and non-focal species were tested to look at the range of focal species DNA that was detectible with the assays. Finally, the DNA copy number measurements were compared with length-based and laboratory-measured carbon biomass of each species to assess the accuracy of using qPCR to estimate copepod naupliar biomass from mixed field samples.

Data Processing Description

Data Processing:
Light Cycler 96 software (Roche, v. 1.1.0.1320)
R (R core team, 2017), packages stats (base R) and car (Fox and Weisberg 2019)

BCO-DMO Processing:
- renamed fields to comply with BCO-DMO naming conventions.

Data Files

File
field_validation.csv (Comma Separated Values (.csv), 4.64 KB) MD5:5956affa03750d932c07ce7e8f261604 Primary data file for dataset ID 869225

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1255697

Deployments

None available yet

Instruments

Automated DNA Sequencer

Supplied Name: Applied Biosystems 3730XL

Supplied Description:

Applied Biosystems 3730XL, with BigDye terminator chemistry v3.1, was used for sequencing species and qPCR products.

Instrument Type

Generic Name: Automated DNA Sequencer

Acronym: Automated Sequencer

Generic Description:

General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.

Elemental Analyzer

Supplied Name: Exeter Analytical CE 440

Supplied Description:

Exeter Analytical CE 440 was used for CHN analysis of copepods.

Instrument Type

Generic Name: Elemental Analyzer

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB01/

Generic Description:

Instruments that quantify carbon, nitrogen and sometimes other elements by combusting the sample at very high temperature and assaying the resulting gaseous oxides. Usually used for samples including organic material.

Fluorometer

Supplied Name: Qubit Fluorometer (Invitrogen)

Supplied Description:

A Qubit Fluorometer (Invitrogen) with the HS Assay was used for measurement of total DNA.

Instrument Type

Generic Name: Fluorometer

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/113/

Generic Description:

A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. The instrument is designed to measure the amount of stimulated electromagnetic radiation produced by pulses of electromagnetic radiation emitted into a water sample or in situ.

Microscope - Optical

Supplied Name: Olympus dissection microscope (model SZX16)

Supplied Description:

An Olympus dissection microscope (model SZX16) fitted with Luminera Infinity-3 was used for sorting and sizing copepod nauplii.

Instrument Type

Generic Name: Microscope - Optical

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/

Generic Description:

Instruments that generate enlarged images of samples using the phenomena of reflection and absorption of visible light. Includes conventional and inverted instruments. Also called a "light microscope".

qPCR Thermal Cycler

Supplied Name: Light Cycler 96 Quantitative PCR Thermalcycler (Roche)

Supplied Description:

A light Cycler 96 Quantitative PCR Thermalcycler (Roche) was used for qPCR measurements.

Instrument Type

Generic Name: qPCR Thermal Cycler

Acronym: qPCR

Generic Description:

An instrument for quantitative polymerase chain reaction (qPCR), also known as real-time polymerase chain reaction (Real-Time PCR).

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
Experiment	Experiment ID	unitless	exp_id
Sample_Name	SampleID used in study	unitless	sample
SizeFraction	Size fraction, lower range	micrometers (um)	no_bcodmo_term
Species	Focal species of assay	unitless	no_bcodmo_term
Cq_Mean	Cycle threshold in qPCR, mean	unitless	no_bcodmo_term
Cq_Error	Standard deviaion of Cycle Threshold	unitless	no_bcodmo_term
MeanCopies	Mean copies (sample-1 m-3) measured in qPCR	mean copies per sample per cubic meter	no_bcodmo_term
SD_copies	SD copies	mean copies per sample per cubic meter	no_bcodmo_term
CalNaupAbundance	Calanoid naupliar abundance	abundance per cubic meter	abundance
CycNaupAbundance	Cyclopoid naupliar abundance	abundance per cubic meter	abundance
CalJuveAbundance	Calanoid copepodite abundance	abundance per cubic meter	abundance
CycJuveAbundance	Cyclopoid copepodite abundance	abundance per cubic meter	abundance

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Database

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Dataset: Quantitative PCR: Field Validation