Today Grace and I did an assessment of oyster survival and growth at Westcott Shellfish. This assessment is our fall time point. We will assess again in the spring.

December goals and daily notebook entries.

This post lists all my ongoing projects and to-do list.

Mcap 2020

Final manuscript

Assessment of oyster survival and growth at Goose Point oysters. This assessment is our fall time point. We will assess again in the spring.

This post details weekly oyster maintenance protocols for the tanks in FTR.

This post details processing of Hawaii 2023 project samples for larval size and symbiont cell density.

Today I went to Point Whitney to sample seed from our PolyIC project and check on our other oysters at Point Whitney.

Today I moved all the oysters in FTR into plastic crates to organize and give the oysters more water flow.

Today I ran redundancy (RDA) and variance partioning analyses to examine environmental drivers of physiology across seasons for the E5 Coral Project. he GitHub repository for this paper can be found here. Check out the README for this project in the GitHub repository for more information.

Today we talked with Neil Thompson at USDA to plan seed transfers for 2025.

During my visit to URI this past week I processed DNA for Sanger sequencing for species identification. These samples are from the 2023 Moorea Symbiotic Exchange project. In this project, I collected corals of three genera (Acropora, Pocillopora, Porites) including adults and recruits. We used Sanger sequencing and RFLP to identify species within these genera.

Jill and I optimized lipid and metabolite tissue preparations for the E5 project. Detailed notes are in Jill’s notebook. Jill is now starting preparations of our samples for lipid and metabolite analyses at UW.

November goals and daily notebook entries.

Today we hosted an outreach event with Maritime High School! We hosted ~35 students for several activities. Here is a quick overview of the activities that we did in the lab. We split the students into two groups since it was a large group.

I have listed active manuscripts below and their status along with my writing goals!

This post details analysis of test metabolomic samples for the E5 timeseries molecular project.

October goals and daily notebook entries.

This post details outplanting 10K Seed at Baywater shellfish.

This post details preparations for outplanting 10K Seed at Baywater shellfish.

Today I ran scripts to analyze Efforts B, E, and D survival incubations for oysters in the lab.

Today I sampled oyster larvae at 14 days post fertilization out at Point Whitney following broodstock PolyIC spawning.

Today I sampled oyster larvae at 8 days post fertilization out at Point Whitney following broodstock PolyIC spawning.

This post details analysis of test lipidomic samples for the E5 timeseries molecular project.

Today I sampled larvae (day 3 post fertilization) at Point Whitney.

Today I ran scripts to analyze Effort D survival incubations for oysters in the lab.

Today we did a broodstock immune priming treatment and spawn with oysters at Point Whitney.

Today I generated PC’s for each species in the E5 physiology time series to use for correlations to gene expression.

This post details initial analysis of resazurin metabolic rate measurements in 10K Seed oysters.

This post shows plots from temperature loggers for oyster field sites and outplant sites.

This post details preliminary analysis of lipidomic and metabolomic data for the Hawaii 2023 larval project.

September goals and daily notebook entries.

Field work notebook posts from 17-20 September at Point Whitney.

This post details protocol for data entry for Goose Point oyster growth data from 9/9/2024 and schedule for oyster survival incubations next week.

This post details activities at Goose Point from 9 September 2024 to collect survival and growth data from our June outplants.

August goals and daily notebook entries.

This post details survival analysis from 10K Seed resazurin trial survivorship measurements.

This post details activities at Point Whitney from 8 August 2024.

This post details sampling for 10K seed hardening project at UW.

July goals and daily notebook entries.

This post details activities at Point Whitney from 15 July 2024.

This post details activities at Point Whitney from 10 July 2024.

This post details activities at Point Whitney from 28 June 2024.

This post describes previous protocols for PolyIC immune priming in oysters using emmersion/bath methods. We will use these protocols to design PolyIC exposure in oysters at Point Whitney.

This post details analysis of the Montipora capitata TagSeq data set using a DESeq2 likelihood ratio test approach and subsequent functional enrichment using goseq. I previously found an error in my functional enrichment code that is corrected in this revised analysis.

This post details activities at Goose Point from 25 June 2024 to outplant oysters.

This post details activities at Point Whitney from 24 June 2024 to prepare Goose Point outplants.

June goals and daily notebook entries.

This post details activities at Point Whitney from 19 June 2024.

This post details overview of spawning activities and a general protocol for spawning at large scale at Point Whitney from 13 June 2024.

This post details activities at Point Whitney from 12 June 2024.

Planning for Survivorship measurements

This post details NCBI SRA upload of ITS2 sequences for the Montipora capitata larval thermal tolerance project on June 11 2024.

This post details download and QC for the ITS2 analysis pipeline for 2023 Hawaii Larval Thermal Tolerance project.

This post list upcoming oyster projects and next stages for our reference.

This post details activities at Point Whitney from 4 June 2024 and preparation of oysters for outplanting at Westcott on 7 June 2024.

This post shows an overview of oyster outplanting efforts for this summer.

This post details activities at Point Whitney from 29 May 2024 trip.

May goals and daily notebook entries.

This post details activities at Point Whitney from 22 May 2024 trip.

This post details activities at Point Whitney from 16 May 2024 trip.

This post details transfer of raw files for sequencing projects to hputnam KITT folder on Andromeda as an additional backup.

This post details ploidy results from Manchester (Pacific Hybreed).

This post details DESeq2 analysis to identify differentially expressed genes for the Montipora capitata 2023 larval thermal tolerance project RNAseq. See my notebook posts and my GitHub repo for information on this project.

This post details activities at Point Whitney from 6 May 2024 trip.

This post details activities at Point Whitney from 1 May 2024 trip.

This post details alignment and assembly steps of the bioinformatic pipeline for the Montipora capitata 2023 larval thermal tolerance project RNAseq. See my notebook posts and my GitHub repo for information on this project.

This post details lipidomics and metabolomics extraction and analysis for samples from the Montipora capitata 2023 larval thermal tolerance project at the Unviersity of Technology Sydney in Australia with Dr. Jennifer Matthews.

April goals and daily notebook entries.

This post details QC of the Montipora capitata 2023 larval thermal tolerance project RNAseq files from second delivery of top off sequencing. See my notebook posts and my GitHub repo for information on this project.

This post details the NCBI Sequence Read Archive upload for my Montipora capitata 2023 larval thermal tolerance project. See my notebook posts and my GitHub repo for information on this project.

This post details activities at Point Whitney from 3 April 2024 trip.

This post details a protocol for KAPA bead clean up of PCR product to remove primer dimers.

This protocol details sample preparation for next-generation sequencing of ITS2 amplicons for identification of Symbiodiniaceae in reef-building coral samples.

This post details protocols for Sanger Sequencing and RFLP assays for species identification of Porites and Pocillopora from gDNA.

The post details ITS2 amplicon PCR amplificaiton and preparation for sequencing.

List of protocols used in the lab at URI for lab-a-thon in March 2024!

March goals and daily notebook entries.

This post details test for subsampling thresholds in the Montipora capitata early life history timeseries dataset. The purpose of these tests is to determine the minimum read depth to detect differences between treatment groups for future sequencing runs.

Prepare data

Load metadata.

#create metadata and load
Metadata<-read.csv("Mcap2020/Data/ITS2/ITS2_Metadata.csv")
rownames(Metadata) <- Metadata$sample_name
Metadata <- Metadata %>%
  select(code, hpf, group, lifestage)
Metadata <- as.matrix(Metadata)
Metadata <- sample_data(data.frame(Metadata))

Load profile level data.

#load data for taxonomy
tax0 <- read_tsv(
  file  = "Mcap2020/Data/ITS2/mcap.profiles.absolute.abund_and_meta.txt",
  n_max = 6) %>%
  dplyr::select(-2) %>% 
  gather(UID, value, -1) %>% 
  spread(1, value) %>%
  clean_names()

tax1 <- as.matrix(tax0[, -1], dimnames = list(tax0$uid, colnames(tax0[-1])))
rownames(tax1) <- tax0$uid
tax <- tax_table(tax1)

#load data for count matrix 
otu0 <- read_tsv(
  file  = "Mcap2020/Data/ITS2/mcap.profiles.absolute.abund_and_meta.txt", col_names = TRUE) 

list<-c("UID", "sample_name", "8", "9")
colnames(otu0)<-list

otu0<-otu0%>%
  select(-1) %>%
  dplyr::slice(7:n()) %>%
  mutate_at(2:ncol(.), as.numeric)

otu1 <- as.matrix(otu0[, -1])
rownames(otu1) <- otu0$sample_name
otu <- otu_table(otu1, taxa_are_rows = FALSE)

#combine to phyloseq object
coral <- phyloseq(otu, tax, Metadata)
coral

Note that taxa 8 is Clade C and taxa 9 is Clade D.

Next load the data for coral strain level information.

#gather the taxonomy names  
taxnames <- read_tsv(
  file  = "Mcap2020/Data/ITS2/mcap.seqs.absolute.abund_only.txt",
  n_max = 0) %>%
  select(-1) %>%
  names(.)

#extract clade letter  
tax0 <- data_frame(
  DIV = taxnames,
  clade = str_extract(DIV, "[A-Z]")
)

tax1 <- as.matrix(tax0)
rownames(tax1) <- tax0$DIV
tax <- tax_table(tax1)

otu0 <- read_tsv(
  file  = "Mcap2020/Data/ITS2/mcap.seqs.absolute.abund_and_meta.txt") %>%
  select(-1, )

otu1 <- as.matrix(otu0[, 37:113])
rownames(otu1) <- otu0$sample_name
otu <- otu_table(otu1, taxa_are_rows = FALSE)

coralDIV <- phyloseq(otu, tax, Metadata)
coralDIV

Now save the object.

save(coral, coralDIV, file = "Mcap2020/Data/ITS2/ITS2_phyloseq.RData")

Build DIV Data

Load the data.

load("Mcap2020/Data/ITS2/ITS2_phyloseq.RData")

coralDIV

View the number of sequence counts in our dataset.

Samps<-read_tsv(file  = "Mcap2020/Data/ITS2/mcap.seqs.absolute.abund_and_meta.txt")

hist(Samps$post_taxa_id_absolute_symbiodiniaceae_seqs, breaks=30)

Samps$post_taxa_id_absolute_symbiodiniaceae_seqs

We have >10,551 sequences for all of our samples.

Generate tree.

random_tree = rtree(ntaxa(coralDIV), rooted=TRUE, tip.label=taxa_names(coralDIV))

Merge phyloseq data.

phylo_coral_DIV = merge_phyloseq(coralDIV, Metadata, random_tree)
phylo_coral_DIV

Filter out samples with low numbers in groups (n=1 per group) from phyloseq object.

phylo_coral_DIV<-ps_filter(phylo_coral_DIV, lifestage != "Metamorphosed Polyp (183 hpf)")
phylo_coral_DIV<-ps_arrange(phylo_coral_DIV, lifestage)

Reorder lifestage.

levels(as.factor(sample_data(phylo_coral_DIV)$lifestage))

sample_data(phylo_coral_DIV)$lifestage = factor(sample_data(phylo_coral_DIV)$lifestage, levels = c("Egg (1 hpf)","Embryo (5 hpf)","Embryo (38 hpf)","Embryo (65 hpf)", "Larvae (93 hpf)", "Larvae (163 hpf)", "Larvae (231 hpf)", "Metamorphosed Polyp (231 hpf)", "Attached Recruit (183 hpf)", "Attached Recruit (231 hpf)"))

levels(as.factor(sample_data(phylo_coral_DIV)$lifestage))

Assign object to a shorter name.

GP1 = phylo_coral_DIV
GP1

Display sample sequencing depth.

min(sample_sums(GP1))

We will attempt subsampling at the minimum count (10,551 sequences) and in steps decreasing from 10551, 8000, 6000, 4000, 2000, 1000, 500.

DIV level analysis

This post details protocls and results from identifying species of Porites and Pocillopora samples for the E5 RoL project.

This post describes correcting code and revising analyses for Montipora capitata 2020 developmental time series project.

This post details seed exposures at Point Whitney on 3/19.

This post details the process I used to apply for a CITES export permit with the US Fish and Wildlife management authority.

This post contains my notes and thoughts from attending the Washington Sea Grant Conference for Shellfish Growers on March 11-12 2024.

This post details Analysis of Variance Simultaneous Component Analyses (ASCA) for the CEABIGR project.

This post details field day and project design on 5 March 2024 at Point Whitney.

This post details sampling on 28 February 2024 at Point Whitney.

Today I played around with another attempt at WGCNA analyses to look at exon expression data for the ceabigr project.

This post details preparations for oyster sampling for Lifestage Carryover project.

February goals and daily notebook entries.

This post details initial QC of the Montipora capitata 2023 larval thermal tolerance project RNAseq files. See my notebook posts and my GitHub repo for information on this project. Also see my notebook post on sequence download and SRA upload for these files.

This post details the NCBI Sequence Read Archive upload for my Montipora capitata 2023 larval thermal tolerance project. See my notebook posts and my GitHub repo for information on this project.

Post detailing preliminary analysis of oyster growth data in the USDA/WSG lifestage carry over project.

Hatchery day at Point Whitney for the USDA & WSG Lifestage Carryover Project.

Today I played around with WGCNA analyses to look at exon expression data for the ceabigr project.

Post detailing meeting notes and design considerations for USDA and WSG projects from 2/5/2024.

THis post details updates to the E5 time series analysis. The GitHub repository for this paper can be found here. Check out the README for this project in the GitHub repository.

This post details experimental design thoughts and brainstorming for the WSG and USDA project efforts.

Hatchery day at Point Whitney for the USDA & WSG projects.

January goals and daily notebook entries.

This post details daily entries for the Fall 2023 Moorea Field Expedition.

December goals and daily notebook entries.

October goals and daily notebook entries.

Genes-SNPs-methylation correlation first attempt for CEABIGR project

================

September goals and daily notebook entries.

Today, we moved larvae that were spawned and reared at Pt. Whitney hatchery (~48 hrs old) into HUDL systemsfor the Roberts Lab USDA oyster project.

August goals and daily notebook entries.

Today, Steven and I went to Point Whitney to trial strip spawning and fertilization for the Roberts Lab USDA oyster project.

This post details respirometry data analysis of the Montipora capitata F1F2 project at the Hawaii Institute of Marine Biology (HIMB) in June 2023.

July goals and daily notebook entries.

This post includes the full notebook for June-July 2023 Montipora capitata coral spawning and field expedition in Hawaii at the Hawaii Institute of Marine Biology. These notebook posts by A. Huffmyer are updated in order of most recent to oldest.

This protocol details the step-by-step use of PreSens/Loligo Microplate respiration system for coral larvae.

Introduction to Sanger sequencing and training for coral species identification.

June goals and daily notebook entries.

This post details WGCNA preliminary analysis for the E5 Deep Dive Project.

May goals and daily notebook entries.

This post details the initial stable isotope metabolomics analysis for the Montipora capitata 2021 larval temperature exposure.

April goals and daily notebook entries.

This post details revisions to calculations for multivariate physiological plasticity analysis for the E5 Rules of Life project.

This post details analysis and bioinformatic steps to generate a gene count matrix from RNAseq data for the E5 Deep Dive Project.

This post details analysis of survival, settlement, physiology, and environmental data for the 2021 Montipora capitata larval temperature project.

March goals and daily notebook entries.

Workshop: Software Carpentry in R - Terminal, GitHub, and R

February goals and daily notebook entries.

Today, I attended a workshop through the UW eScience Data Science postdoc group for an introduction to DVC, a version-control platform.

This post is a running log of miscellaneous coding and TidyTuesday exercises!

January goals and daily notebook entries.

This post details integrative analysis and plotting for response variables in the Montipora capitata 2020 developmental time series.

This post details re-analyzing TagSeq data for the 2020 M. capitata developmental timeseries project to a more recent v3 genome with annotation.

This post shows newly revised figures and revisions to analysis for the 2020 Montipora capitata development project.

This post details recent updates to the Montipora capitata developmental time series 16S analysis.

Writing

1. Revise Montipora capitata 2020 time series figures
   • Conceptual
   • Metabolomic
   • Gene Expression
   • Physiology
   • Integration
2. Revise Montipora capitata 2020 time series text to PNAS short format
3. Work with Emma to revise results of E5 time series manuscript
4. Reanalyze TagSeq data to new M. capitata genome

This post details analysis of respirometry data for the Acropora pulchra larval metabolism experiment in Moorea in Oct 2022.

This post details alignment of TagSeq sequences from the Mcap2020 developmental time series project to the symbiont genome. We will evaluate the results of this alignment to see if we have enough sequences to analyze symbiont-specific gene expression.

This post details environmental measurements analysis for the 2022 Acropora larval metabolism experiment from the Moorea 2022 Field Expedition.

This post details the NCBI Sequence Read Archive upload for Ariana Huffmyer’s Montipora capitata 2020 early life history timeseries project.

This post includes the full notebook for Sept-Oct 2022 Acropora coral spawning and field expedition in Moorea, French Polynesia. These notebook posts by A. Huffmyer are updated in order of most recent to oldest.

Writing

1. Full draft of E5 results section for the 2020 time series project
2. Submit proofs for Pocillopora acuta diversity paper~
3. Revise Montipora capitata 2020 time series metabolomics and gene expression figures and associated text

Writing

1. First draft of E5 time series results section and outline of discussion section
2. Submit proofs for science education paper
3. Start Montipora capitata 2020 time series revisions

Writing

1. First draft of E5 time series results section and outline of discussion section
2. Submit revision for Pocillopora acuta genetic diversity paper
3. Go over Montipora capitata 2020 time series revisions with HP
4. Start Montipora capitata 2020 time series revisions

This post details multivariate physiological plasticity analysis for the E5 Rules of Life project.

Writing

1. Finish and send final draft of Montipora capitata ontogeny 2020 manuscript to co-authors
2. First draft of E5 time series manuscript by end of August with full results and outline of discussion section
3. Submit final revision for science education manuscript
4. Write methods and results section of Montipora capitata larval temperature exposure 2021 project
5. Submit revision for Pocillopora acuta genetic diversity paper

This protocol details the methods for collecting Montipora capitata bundles during spawning, fertilizing gametes, and rearing and settling larvae in Hawai’i during the summer months.

This protocol details the step-by-step process for incubations for stable isotope metabolomics for coral larvae.

Writing

1. Write results of Montipora capitata development manuscript
2. Draft results of E5 time series manuscript

Writing

1. Methods and results of Montipora capitata development manuscript
2. Submit final revision of science identity manuscript (delayed until August 2022)

This post details preliminary analyses of multi-omic datasets collected in the Montipora capitata early life history time series project.

This protocol describes the process for taking photographs of preserved coral larvae for measurement of larval size (volume, surface area, length, etc.).

This protocol details symbiont cell counts in coral larval tissue samples. This protocol is modified from the E5 cell counts protocol.

This protocol details metabolite extraction from coral larval samples labeled with stable isotopes (C13 bicarbonate) and control samples (C12 bicarbonate).

This protocol details quantification of soluble protein from coral larval samples. This protocol is modified from the E5 Protein Protocol.

This post details sample processing completed in April 2022 for the 2021 M. capitata larval thermal exposure experiment conducted in Hawaii.

This protocol details chlorophyll measurement in coral larval tissue samples. This protocol is modified from the E5 chlorophyll protocol.

This protocol details quantification of carbohydrate content in coral larvae in 96-well plates modified from the Bove Baumann protocol and the Putnam Lab carbohydrate protocol.

This post details 16S analysis of the Putnam Lab oyster project using the Mothur pipeline based on A. Huffmyer’s pipeline here.

Writing

1. Write results of Montipora capitata development manuscript
2. Start discussion of Montipora capitata development manuscript

This post details analysis of TagSeq data from previous alignment to M. capitata version 1 assembly and version 2 assembly.

Writing

1. Finish methods of Montipora capitata development manuscript
2. Write results of Montipora capitata development manuscript
3. Start discussion of Montipora capitata development manuscript

This post details QC and filtering steps that were done in both Mothur and QIIME2 to compare the sequence QC results.

This post details analyzing relative abundance and diversity metrics in R output from mothur analysis of 16S data for the 2020 early life history Montipora capitata time series.

This post details continued analysis of metabolomics data for the 2020 M. capitata developmental time series with WGCNA.

This post details 16S data analysis using the Mothur pipeline for the 2020 Montipora capitata developmental time series.

Publications

1. Revise and submit intraspecific diversity paper
2. Write methods for Pocillopora acuta thermal exposure paper
3. Outline revisions for Life History energetics perspective paper
4. Outline results section of E5 timeseries manuscript
5. Finish methods and write results of Montipora capitata development manuscript

This post details QC and QIIME analysis for the 16S analysis adapted from the pipeline developed by Emma Strand.

This post details SymPortal analysis for the ITS2 analysis pipeline modified from Kevin Wong pipeline.

This post details MultiQC for the ITS2 analysis pipeline. 16S files are analyzed in a separate post.

This post details QC and QIIME analysis for the 16S analysis adapted from the pipeline developed by Emma Strand.

This post details MultiQC for the 16S analysis pipeline adapted from the pipeline developed by Emma Strand. In this post there is information for moving/managing ITS2 sequences, but this post details preliminary analysis of 16S. ITS2 files will be analyzed in a separate post.

This post details all activities in Moorea in December 2021 for Symbiont Integration project for the study species Pocillopora acuta.

This posts describes extraction protocols for ITS2 and 16s sequencing for the 2020 Montipora capitata developmental time series.

Publications

1. Revise and submit intraspecific diversity paper
2. Finish revisions and submit larval thermal conditioning paper
3. Outline revisions for Life History energetics perspective paper

Environmental data analysis for the E5 Rules of Life Project continued.

This posts describes the QC process from November 2021 for the E5 Rules of Life timeseries data.

This post continues analysis of environmental time series data for the E5 Rules of Life Project.

This post details environmental time series QC and plotting for the E5 Rules of Life project.

This posts describes weighted gene co-expression analysis (WGCNA) applied to metabolmoics data from the 2020 Montipora capitata early life history developmental time series.

Proposals and applications

1. Submit education paper revisions to CBE - Life Sciences Education (November 9)
2. Submit Science Incubator proposal (November 10)
3. Submit NSF OCE proposal (November 12)
4. Prepare E5 time series talk for Moorea LTER Annual Meeting in early December

Post describing Pocillopora larval collection for collaborative projects with Carneige and Rutgers in June 2021.

Post detailing larval settlement and maintenance in June 2021 for M. capitata spawning.

Post detailing stable isotope time series data collection for methods optimization and development for coral larvae in collaboration with Rutgers University.

Post detailing temperature exposure and experimental design for M. capitata larval temperature experiment in June 2021.

Post detailing survival measurements and experimental maintenance in larval temperature experiment with M. capitata in June 2021.

Collection, fertilization, and rearing of Montipora capitata spawned gametes during the June 2021 spawning at the Hawaii Institute of Marine Biology.

Post details sampling and data collection for M. capitata larval temperature exposure experiment in June 2021.

Post detailing tank and experimental set up for Montipora capitata coral spawning in Hawaii in June 2021.

Post detailing culture maintenance of embryos of Montipora capitata during June 2021 larval temperature experiment and spawning.

Post detailing sampling of Mcapitata larvae collected as baseline samples prior to temperature experiment in June 2021.

This entry details sample processing for Mcapitata 2020 early life history samples for metabolomics, larval size and symbiont cell densitites, and RNA excxtraction for TagSeq.

This entry describes the sampling and respirometry data collection for Acropora hyacinthus in Moorea, French Polynesia at CRIOBE and Gump Research Stations.

This entry lists the runs and data collection for early life history respirometry (photosynthesis and respiration) in early life stages (larvae, recruits, and juveniles) of Montipora capitata in July 2020.

This notebook entry details methods for larval rearing and settlement for Montipora capitata early life history in July 2020. Sampling of these life stages is described in additional notebook entries.

This entry details the sampling and collection of embryos, larvae, and recruits of Montipora capitata during July-Aug 2020 spawning period.

This entry describes the protocol for measuring photosynthesis and respiration in early life history stages of Montipora capitata.

This protocol provides an overview of sampling early life history stages for Montipora capitata in July-August 2020.

Collection, fertilization, and rearing of Montipora capitata spawned gametes during the July 2020 spawning at the Hawaii Institute of Marine Biology in collaboration with C. Drury and J. Hancock in the Gates Coral Lab.