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