Dataset: Neocalanus flemingeri incubation experiment: gene expression

Name: Gene expression profiles for Neocalanus flemingeri pre adults (CV) from the M/V Dora in the Gulf of Alaska station GAK1 on 2019-04-15
Published: 2024-04-01
Keywords: Gulf of Alaska, Neocalanus flemingeri, incubation experiment, lipid accumulation, gene expression, oceans

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Cite This Dataset

Spatial Extent: N:59.8443 E:-149.4838 S:59.8443 W:-149.4838

Gulf of Alaska

Temporal Extent: 2019-04-15

Project:

Collaborative Research: Molecular profiling of the ecophysiology of dormancy induction in calanid copepods of the Northern Gulf of Alaska LTER site (Diapause preparation)

Principal Investigator:

Petra H. Lenz (University of Hawaii at Manoa, PBRC)

Scientist:

Daniel K. Hartline (University of Hawaii at Manoa, PBRC)

Vittoria Roncalli (University of Hawaii at Manoa, PBRC)

Student:

Lauren N Block (University of Hawaii at Manoa, PBRC)

Contact:

Petra H. Lenz (University of Hawaii at Manoa, PBRC)

Data Manager:

Matthew C. Cieslak (University of Hawaii at Manoa, PBRC)

BCO-DMO Data Manager:

Lynne M. Merchant (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2024-04-01

Restricted:

Validated:

Current State:

Preliminary and in progress

Gene expression profiles for Neocalanus flemingeri pre adults (CV) from the M/V Dora in the Gulf of Alaska station GAK1 on 2019-04-15

Abstract:

This experimental dataset includes relative expression of individual Neocalanus flemingeri stage CV individuals incubated for different lengths of time and four different food treatments. The experimental protocol and results are described in detail in Roncalli et al., 2023. Briefly, field-collected N. flemingeri were allowed to molt into stage CV and then sorted into four different treatments: no food, low carbon, high carbon and high carbon with diatoms. After a one-week incubation, individuals from all four treatments were processed individually for RNA-Seq. In addition, following two and three-week incubations, copepods from the three fed treatments were processed individually for RNA-Seq. Short-sequence reads were mapped against a reference transcriptome and normalized gene expression was computed for each transcript. The dataset includes log-transformed relative gene expression in reads per kilo base per million reads (RPKM) (log2[RPKM+1]). The dataset also includes the list of differentially expressed genes and a look-up table that cross-references the hierarchical identifications of transcripts generated by the Trinity assembly software and the corresponding National Center for Biotechnology Information (NCBI) accession number. These data are further described in the following publications: Roncalli, et al. (2023) (DOI: 10.1093/plankt/fbad045) and Roncalli, et al. (2019) (DOI: 10.1038/s42003-019-0565-5)

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Description

These data are further described in the following publications:

Roncalli, V., Block, L. N., Niestroy, J. L., Cieslak, M. C., Castelfranco, A. M., Hartline, D. K., & Lenz, P. H. (2023). Experimental analysis of development, lipid accumulation and gene expression in a high-latitude marine copepod. Journal of Plankton Research, 45(6), 885–898. https://doi.org/10.1093/plankt/fbad045

Roncalli, V., Cieslak, M. C., Germano, M., Hopcroft, R. R., & Lenz, P. H. (2019). Regional heterogeneity impacts gene expression in the subarctic zooplankter Neocalanus flemingeri in the northern Gulf of Alaska. Communications Biology, 2(1). https://doi.org/10.1038/s42003-019-0565-5

Methods & Sampling

Zooplankton were collected on a day-trip to station GAK1 (59º50.7′ N, Long: 149º28′ W, depth 264 m, Gulf of Alaska) (http://research.cfos.uaf.edu/gak1/) aboard the M/V Dora on April 15, 2019. Collections were made using QuadNet with two 150 µm and two 53 µm mesh nets towed vertically from 100 to 0 m. Collection details are provided in Roncalli et al. (2023). Zooplankton samples were diluted, brought back to the laboratory and sorted under a dissection microscope to select stage CIV Neocalanus flemingeri individuals. As individuals molted into CVs, they were removed from the holding containers and transferred into 750 ml Falcon flasks with 3 individuals per flask and assigned to one of the 4 food treatments, as described in detail in Roncalli et al. (2023). Three individuals were preserved upon molting (Wk0). Individuals were harvested at 3 incubation times (Wk1, Wk2 and Wk3) and preserved in RNALater Stabilization Reagent. Preserved copepods were frozen first in -40ºC during the experiment, and then transferred to −80°C until further processing.

Total RNA extraction, library construction, RNA sequencing and quality control: total RNA was extracted from individuals using QIAGEN RNeasy Plus Mini Kit (catalog # 74134) in combination with a Qiashredder column (catalog # 79654). Sequencing was performed on 3 Wk0 individuals and 3 replicate individuals for each time x treatment combination. Total RNA was shipped on dry ice to the Georgia Genomics Bioinformatics Core (https://dna.uga.edu) for RNA-Seq. There, double-stranded cDNA libraries (KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads (cat #KK8421]) from each individual were multiplexed and sequenced using an Illumina Next-Seq 500 instrument (High-Output Flow Cell, 75 bp, paired end). Quality of each RNA-Seq library was reviewed with the FastQC software28. From each RNA-Seq library, low quality reads were removed using FASTQ Toolkit (v. 2.2.5 within BaseSpace). Illumina adaptors, reads <50 bp long, reads with an average Phred score <30 and the first 12 bp from each read, were removed from each library.

Data Processing Description

Ribosomal RNA was removed from each RNA-Seq library (SortMeRNA) (Kopylova et al., 2012) prior to mapping reads to a standard N. flemingeri reference transcriptome (NCBI: BioProject PRJNA496596, TSA GHLB01000000) (Roncalli et al., 2019). Reads were mapped against the reference using kallisto software (default settings; v.0.43.1) (Bray et al., 2016) and Bowtie2 software(v2.3.5.1) (Langmead et al., 2009). Counts generated by the Bowtie2 mapping, were normalized using the RPKM method (reads per kilobase of transcript length per million mapped reads) (Mortazavi et al., 2008), followed by log2 transformation of the relative expression data (Log2[RPKM+1]).

For gene expression analysis, kallisto-mapped transcripts with low expression (< 1 count per million in all treatments [1cpm]) were removed leaving 46,416 transcripts (90%) that were tested for differential gene expression using the generalized linear model (Bioconductor package EdgeR, R v. 3.12.1) with p-values were adjusted for false discovery rate (FDR) using the Benjamini-Hochberg correction (default algorithm weight01) (Robinson et al., 2010).

BCO-DMO Processing

Steps for processing the main dataset file, the differentially expressed genes file, and the Seq ID and GenBank accession cross reference table.

1) Rename parameters in the cross reference file from Trinity_ID to seq_id and Genbank_Accession_number to Genbank_accession
2) Add the version 1 to the Genbank_accession values that weren't provided in the submitted file.
3) Join the cross reference file with the differentially expressed genes file to add the corresponding Genbank accession numbers to seq_id values.
4) Join the cross reference file with the relative gene expression file to add the corresponding Genbank accession numbers to seq_id values.
5) Add metadata from a submitted metadata file.
6) Add a date field that is created from day, month, and year metadata parameters and of the format %Y-%m-%d.
7) Add a NCBI URL for each Genbank accession number to the cross reference file, the differentially expressed genes file, and the relative gene expression file.
8) Reorder the columns to move the metadata columns to the front of the relative gene expression file
9) Rename parameters to follow the BCO-DMO naming protocol. Rename column headers that have a period or space in their name to an underscore. Remove (m) from the Depth range parameter since it will be indicated in the parameters section of the dataset page.

Steps to create an unpivoted version of the submitted relative gene expression file and a metadata table

1) Combine the submitted metadata table and a BCO-DMO data manager created metadata table (week_after_molting_to_CV, feeding_protocol, BioProject, BioSample) into one metadata table.
2) Rename column headers in the metadata table according to BCO-DMO naming protocols. Replace spaces with underscores and remove (m) from the Depth range parameter name since this unit will be included in the units section of parameter definitions on the dataset page.
3) In the unpivoted file, rename Genbank_Accession_number to Genbank_accession.
4) Add a GenBank accession URL
5) Create a date field of the format %Y-%m-%d from the month, day, and year columns where the month is padded with 0.
6) Unpivot the submitted relative gene expression file to transform the source seq_id and Replicate columns into columns of seq ID, replicate, and relative gene expression
7) Rename the replicate values to replace the period with an underscore. This is to match the replicate naming in the final relative gene expression dataset file.
8) Join the combined metadata table with the unpivoted file to add the metadata to the unpivoted file
9) Remove experiment descriptions and NCBI metadata fields, except GenBank accession numbers and urls, from the unpivoted file to reduce file size.
10) Join a submitted cross reference table of seq_id to Genbank accession values with the unpivoted file on seq_id to add the Genbank accession values
11) Append ‘.1’ to the Genbank accession values since the Genbank accession number includes the version, and for this data, it is version 1.
12) Reorder the columns of the combined metadata file to move NCBI accession values to the end
13) Reorder columns to move metadata columns to the front of the unpivoted file.

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1756767
NSF Division of Ocean Sciences	OCE-1756859

Deployments

Deployment	Synonyms	Start Date	Platform	Investigator
Lenz_Gulf_of_Alaska_2019-04-15		2019-04-15	M/V Dora	Petra H. Lenz (Principal Investigator)	data info

Instruments

Automated DNA Sequencer

Supplied Name: Illumina Next-Seq 500

Supplied Description:

Desktop sequencer

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.

Microscope - Optical

Supplied Name: Dissection microscope

Supplied Description:

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".

Plankton Net

Supplied Name: QuadNet

Supplied Description:

Two 150 µm and two 53 µm mesh nets

Instrument Type

Generic Name: Plankton Net

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/22/

Generic Description:

A Plankton Net is a generic term for a sampling net that is used to collect plankton. It is used only when detailed instrument documentation is not available.

Parameters

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

Supplied Name	Supplied description	Supplied Units	Standard Name
seq_id	Sequence identification using Trinity identification of assembled transcripts	unitless	sequence
Genbank_accession	NCBI GenBank acession number	unitless	Genbank_accession
Genbank_accession_url	NCBI GenBank acession URL	unitless	external_link
Organism_Name	Species analyzed	unitless	species
Station	Station	unitless	station
Latitude	Latitude. Locations south of equator are negative.	decimal degrees	lat
Longitude	Longitude. Locations west of prime meridian are negative.	decimal degrees	lon
Collection_date	Collection date Format: %Y-%m-%d	unitless	date
Year	Collection year	unitless	year
Month	Collection month	unitless	month
Day	Collection day	unitless	day
Depth_range	Collection depth range	meters (m)	depth_r
T0_1	Relative gene expression for replicate T0_1. Food protocol:: No food, Week after molting to CV: 0	Log2[RPKM+1]	no_bcodmo_term
T0_2	Relative gene expression for replicate T0_2. Food protocol:: No food, Week after molting to CV: 0	Log2[RPKM+1]	no_bcodmo_term
T0_3	Relative gene expression for replicate T0_3. Food protocol:: No food, Week after molting to CV: 0	Log2[RPKM+1]	no_bcodmo_term
NF_1	Relative gene expression for replicate NF_1. Food protocol:: No food, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
NF_2	Relative gene expression for replicate NF_2. Food protocol:: No food, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
NF_3	Relative gene expression for replicate NF_3. Food protocol:: No food, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
BW1_1	Relative gene expression for replicate BW1_1. Food protocol:: Low Carbon diet, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
BW1_2	Relative gene expression for replicate BW1_2. Food protocol:: Low Carbon diet, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
BW1_3	Relative gene expression for replicate BW1_3. Food protocol:: Low Carbon diet, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
GW1_1	Relative gene expression for replicate GW1_1. Food protocol::High Carbon diet, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
GW1_2	Relative gene expression for replicate GW1_2. Food protocol::High Carbon diet, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
GW1_3	Relative gene expression for replicate GW1_3. Food protocol::High Carbon diet, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
YW1_1	Relative gene expression for replicate YW1_1. Food protocol::High Carbon diet + diatom, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
YW1_2	Relative gene expression for replicate YW1_2. Food protocol::High Carbon diet + diatom, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
YW1_3	Relative gene expression for replicate YW1_3. Food protocol::High Carbon diet + diatom, Week after molting to CV: 1	Log2[RPKM+1]	no_bcodmo_term
BW2_1	Relative gene expression for replicate BW2_1. Food protocol::Low Carbon diet, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
BW2_2	Relative gene expression for replicate BW2_2. Food protocol::Low Carbon diet, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
BW2_3	Relative gene expression for replicate BW2_3. Food protocol::Low Carbon diet, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
GW2_1	Relative gene expression for replicate GW2_1. Food protocol::High Carbon diet, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
GW2_2	Relative gene expression for replicate GW2_2. Food protocol::High Carbon diet, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
GW2_3	Relative gene expression for replicate GW2_3. Food protocol::High Carbon diet, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
YW2_1	Relative gene expression for replicate YW2_1. Food protocol::High Carbon diet + diatom, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
YW2_2	Relative gene expression for replicate YW2_2. Food protocol::High Carbon diet + diatom, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
YW2_3	Relative gene expression for replicate YW2_3. Food protocol::High Carbon diet + diatom, Week after molting to CV: 2	Log2[RPKM+1]	no_bcodmo_term
BW3_1	Relative gene expression for replicate BW3_1. Food protocol: Low Carbon diet, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term
BW3_2	Relative gene expression for replicate BW3_2. Food protocol: Low Carbon diet, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term
BW3_3	Relative gene expression for replicate BW3_3. Food protocol: Low Carbon diet, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term
GW3_1	Relative gene expression for replicate GW3_1. Food protocol: High Carbon diet, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term
GW3_2	Relative gene expression for replicate GW3_2. Food protocol: High Carbon diet, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term
GW3_3	Relative gene expression for replicate GW3_3. Food protocol: High Carbon diet, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term
YW3_1	Relative gene expression for replicate YW3_1. Food protocol: High Carbon diet + diatom, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term
YW3_2	Relative gene expression for replicate YW3_2. Food protocol: High Carbon diet + diatom, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term
YW3_3	Relative gene expression for replicate YW3_3. Food protocol: High Carbon diet + diatom, Week after molting to CV: 3	Log2[RPKM+1]	no_bcodmo_term

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Dataset: Neocalanus flemingeri incubation experiment: gene expression