Master’s Thesis Committee Meeting #3

Progress check-in on analyses

Sarah Tanja

2025-05-06

MS Thesis Proposal

proposal is complete
submitted signed proposal pdf to SAFS milestones

Clarifications on experiment

12 treatments across leachate exposure and developmental milestones (hpf)
Microscopy n=9
July 5th & 6th
Originally n=10, dropped 1 cross due to no fertilization
Microbiome n=5 & Transcriptomics n=5
July 5th, 6th & 7th
Originally n=15, pooled samples (3 per) to have enough material to extract

Microscopy

Annotations

7 Control 4, 9, & 14 hpf

Direction of cell division from 2 to 4 proceeds from the inside out

Initial results

Microbiome

Partnered with mim_c for analysis
Shannon diversity non-significant across features

Taxa barplot

Maaslin2 significant differential features

Leachate decreased abundance in key coral microbes

Arcobacteraceae (mixotroph, carbon fixation)
Lactobacillus (beneficial probiotic, carbohydrate fermentation produces lactic acid)
Alteromonas (contribute to carbohydrate degradation, supports nutrient cycling, DMSP (dimethylsulfoniopropionate) metabolism –> tolerance to thermal stress)

Important

PICRUSt2 : infer functional pathway abundances

Transcriptomics

PCA

DEGs

Interaction term

#Set DESeq2 design for 2 factors and their interaction

dds_i <- DESeqDataSetFromMatrix(countData = gcm_tidyfilt,
                                  colData = metadata,
                                  design = ~ embryonic_phase + pvc_leachate_level + embryonic_phase:pvc_leachate_level)

summary(results(dds_i_wald), alpha = 0.05)

out of 22634 with nonzero total read count
adjusted p-value < 0.05
LFC > 0 (up)       : 217, 0.96%
LFC < 0 (down)     : 24, 0.11%
outliers [1]       : 30, 0.13%
low counts [2]     : 4824, 21%
(mean count < 7)

4 hpf has 1 gene with a LFC > 0

dds_4 <- DESeqDataSetFromMatrix(countData = gcm_tidyfilt_4,
                              colData = metadata_4,
                              design = ~ pvc_leachate_level)

summary(results(dds_4_wald), alpha = 0.05)
out of 14191 with nonzero total read count
adjusted p-value < 0.05
LFC > 0 (up)       : 1, 0.007%
LFC < 0 (down)     : 0, 0%
outliers [1]       : 198, 1.4%
low counts [2]     : 0, 0%
(mean count < 2)

9 hpf & 14 hpf have zero pairwise DEGs

Heatmaps

GO functional enrichment

Important

Interaction: need to confirm model inputs for multifactorial model of differential expression
Pairwise: true list of DEG’s very very small… (0, or 1 differentially expressed gene!?)
Currently removing outliers, checking model fit assumptions as per MarineOmics tutorial
We will either have 1 really interesting gene to talk about, or show no differential expression due to leachate
Differential expression between embryonic stages this early has not previously been demonstrated

Remaining timeline

SP25 Finish Analyses (focus on Results section)
June 15th 2025 | deadline to apply for SAFS finishing fellowship for AU25
On-Leave status for SU25 (no funding, GI Bill runs out at the end of SP25)
Writing Results & Discussion SU25
Back again AU25
Sep 20th 2025 Submit Thesis Draft
Nov 20th 2025 Final Exam
Dec 12th 2025 Submit Thesis to UW

Links

coral-embryo-scope

coral-embryo-microbiome

coral-embryo-RNAseq

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