Dataset: Neocalanus flemingeri incubation experiment: gene expression

Name: Gene expression profiles for Neocalanus flemingeri pre adults (CV) exposed to four different experimental food conditions collected from the M/V Dora in the Gulf of Alaska at station GAK1 from April 2019
Published: 2024-05-30
Keywords: Gulf of Alaska, Neocalanus flemingeri, incubation experiment, lipid accumulation, gene expression, oceans

Cite This Dataset

DOI:10.26008/1912/bco-dmo.914459.1

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 Hawaiʻi at Mānoa, PBRC)

Scientist:

Daniel K. Hartline (University of Hawaiʻi at Mānoa, PBRC)

Jeanette L. Niestroy (University of Hawaiʻi at Mānoa, PBRC)

Vittoria Roncalli (University of Hawaiʻi at Mānoa, PBRC)

Student:

Lauren N Block (University of Hawaiʻi at Mānoa, PBRC)

Contact:

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

Technician:

Matthew C. Cieslak (University of Hawaiʻi at Mānoa, PBRC)

Data Manager:

Matthew C. Cieslak (University of Hawaiʻi at Mānoa, PBRC)

BCO-DMO Data Manager:

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

Version:

Version Date:

2024-05-30

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Gene expression profiles for Neocalanus flemingeri pre adults (CV) exposed to four different experimental food conditions collected from the M/V Dora in the Gulf of Alaska at station GAK1 from April 2019

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 kilobase per million reads (RPKM) (log2[RPKM+1]). The dataset also includes a 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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Archival Copy

Version Date	Archive	Persistent Identifier	Date Assigned
2024-05-30	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.26008/1912/bco-dmo.914459.1	2024-07-16

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. Commun 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. Loaded submitted files into the BCO-DMO laminar processor. Submitted files loaded are 2024-Jan-sra_result-Seward2019-Expt.xlsx, File1-GeneExpression-Log2(RPKM+1).csv, File2-DEGs-GLM Analysis.csv, and File3-CrossReference-Trinity Genbank.csv.
2. Renamed parameters in the cross reference file from Trinity_ID to seq_id to match the parameter names in the other files and Genbank_Accession_number to Genbank_accession
3. Added the version number 1, suffix ‘.1’, to the Genbank_accession values of File3-CrossReference-Trinity Genbank.csv because the NCBI GenBank accession numbers should contain a version number.
4. Joined the cross reference file with the differentially expressed genes file, File3-CrossReference-Trinity Genbank.csv, on the column seq_id to add the corresponding GenBank accession numbers to the file.
5. Joined the cross reference file with the relative gene expression file, File1-GeneExpression-Log2(RPKM+1).csv, on the column seq_id to add the corresponding GenBank accession numbers to the file.
6. Joined the submitted metadata table to the relative gene expression file, File1-GeneExpression-Log2(RPKM+1).csv, to add the metadata to the file.
7. Added a date field of the format %Y-%m-%d created from the day, month, and year values.
8. Reordered the columns to move the metadata columns to the front of the relative gene expression file
9. Renamed parameters in the relative gene expression file to follow the BCO-DMO naming protocol. Renamed column headers that have a period or space in their name to an underscore. Removed ‘(m)’ from the Depth range parameter name since units will be indicated in the parameters section of the dataset page.

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

1. Load in submitted files and a data manager metadata file into the BCO-DMO laminar processor.
2. First loaded in the submitted metadata file 2024-Jan-sra_result-Seward2019-Expt.xlsx and the data manager metadata file dm_replicate_experiments_metadata.csv into laminar.

The submitted metadata file has the columns: Experiment Accession, Experiment Title, Organism Name, Year, Month, Day, Station, Latitude, Longitude, Depth range (m), Study Accession, Study Title, Sample Accession, Replicate. The data manager created metadata file has the columns: Replicate, week_after_molting_to_CV, feeding_protocol, BioProject, BioSample
3. Joined the submitted metadata table and the data manager created metadata table on the Replicate field into a new metadata table named metadata_table_with_ncbi_accessions.
4. Renamed the column headers in the new metadata table according to BCO-DMO naming protocols. Replaced spaces with underscores and removed the text “(m)” from the Depth range parameter name since this unit will be included in the units section of parameter definitions on the dataset page.
5. Added a collection date column of the format %Y-%m-%d from the year, month, and day columns.
6. Loaded into laminar the submitted cross reference file named File3-CrossReference-Trinity Genbank.csv.
7. Added the version number text ‘.1’ to the parameter ‘Genbank Accession Number’ in the lookup table.
8. Loaded in the submitted relative gene expression file named “File1-GeneExpression-Log2(RPKM+1).csv”.
9. Applied the laminar process ‘unpivot’ to the relative gene expression file.
10. Unpivoted on the column names which are of the form T0.1, T0.2, NF.2, GW1.3.
11. Named the unpivoted table “unpivoted_relative_gene_expression” to later save as a csv file.
12. In the unpivoted file, renamed Genbank_Accession_number to Genbank_accession.
13. Joined the metadata table “metadata_table_with_ncbi_accessions” with the unpivoted table “unpivoted_relative_gene_expression” on the column “Replicate” to add metadata to the unpivoted table.
14. Joined the file “File3-CrossReference-Trinity Genbank.csv” with the unpivoted file “unpivoted_relative_gene_expression” on the column “Trinity_ID” in the cross-reference file and “seq_id” in the unpivoted file.
15. Because the main dataset will have the Replicate column names in the metadata table “metadata_table_with_ncbi_accessions” were renamed from T0.1 to T0_1, etc., the replicate columns in the joined table were renamed in the same pattern by replacing the period with an underscore so that the final metadata table will match the run_id values in the main dataset file. The same renaming was done for the joined table “unpivoted_relative_gene_expression”.
16. Genbank_Accession_number was renamed to match the pattern of the other accession parameter names.
17. The parameter fields in the unpivoted table and metadata table were reordered to group the accession parameters at the end of the tables.
18. Removed the NCBI accession numbers and titles except for the GenBank accession numbers from the unpivoted file to reduce the file size.

Data Files

File
914459_v1_relative_gene_expression.csv (Comma Separated Values (.csv), 13.06 MB) MD5:05703749ca48ac3358891cb931c99249 Primary data file for dataset ID 914459, version 1 Relative gene expression given in log2(RPKM +1) for all transcripts based on RNA-Seq data mapped against a reference transcriptome.

Supplemental Files

File
Differentially expressed genes filename: differentially_expressed_genes.csv (Comma Separated Values (.csv), 477.77 KB) MD5:c142ca72eca93cbed14437cf5a795cd4 List of differentially expressed genes (DEGs) calculated using EdgeR GLM analysis. Columns: seq_id, Genbank_accession See the parameters section of this dataset for definitions of seq_id and Genbank_accession parameters used in this file. Blank values in Genbank_accession represent no corresponding value for seq_id
Metadata table including NCBI accessions filename: supplemental_files/metadata_table_with_ncbi_accessions.csv (Comma Separated Values (.csv), 10.55 KB) MD5:9f972fafe716cdaf58d5581fac488dd9 See the supplemental file "Metadata table parameter definitions", metadata_table_parameter_definitions.csv, for definitions of parameters included in this metadata table. Columns: Replicate, Organism_Name, Station, Latitude, Longitude, Collection_date, Year, Month, Day, Depth_range, week_after_molting_to_CV, feeding_protocol, BioProject, Study_Accession, Study_Title, Experiment_Accession, Experiment_Title, BioSample, Sample_Accession
Metadata table parameter definitions filename: metadata_table_parameter_definitions.csv (Comma Separated Values (.csv), 1.54 KB) MD5:14b21267df32633e4585862b913cf479 This file contains parameter definitions for the supplemental file "Metadata table including NCBI accessions", metadata_table_with_ncbi_accessions.csv Columns: suppliedName, description, suppliedUnits, no_data_value, bcodmo_standard_parameter_name, Datatype, Format suppliedName = Supplied Name description = parameter description suppliedUnits = parameter units no_data_value = fill value for no data bcodmo_standard_parameter_name = BCO-DMO standard parameter name DataType = parameter data type Format = date format if the parameter data type is date
Seq ID and GenBank accession numbers cross-reference table filename: seq_id_genbank_accession_cross_reference.csv (Comma Separated Values (.csv), 1.96 MB) MD5:65425ff3133194f7defd2090c4789ef0 Cross-reference between Trinity IDs and Genbank accession numbers Columns: seq_id, Genbank_accession See the parameters section of this dataset for definitions of seq_id and Genbank_accession parameters used in this file.
Species WoRMS taxonomy filename: species_list.csv (Comma Separated Values (.csv), 211 bytes) MD5:0996e3c2eab4418bf250f26afec882aa Species WoRMS taxonomy table with columns: ScientificName, AphiaID, LSID, Authority, Class, Order, Family, Genus, Species
Unpivoted version of the relative gene expression dataset filename: supplemental_files/unpivoted_relative_gene_expression.csv (Comma Separated Values (.csv), 230.86 MB) MD5:1fb45c6c56d0b9edafb19491614aa68d See the supplemental file "Metadata table parameter definitions", metadata_table_parameter_definitions.csv, for definitions of parameters included in this file that are not listed as parameters on this dataset page. Columns: seq_id, Genbank_Accession, Organism_Name, Station, Latitude, Longitude, Collection_date, Year, Month, Day, Depth_range, Replicate, relative_expression, week_after_molting_to_CV, feeding_protocol

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
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