Long non-coding RNA discovery in Pocillopora sp.

For a full overview, take a look at my project compendium.

Final Zenodo Release

FISH 546 project repo examining the expression of long non-coding RNAs in corals

Introduction

This project uses RNA-seq data from Danielle Becker's experiment exposing colonies of Pocillopora sp. to low level nutrient conditions (nitrogen and phosphorous).

• Original Experiment GitHub Repo
• Project OSF Link

Experimental Design

This study attempts to identify and understand the expression of long non-coding RNAs in corals experiencing low level nutrient enrichment. Long non-coding RNAs (lncRNAs), RNA strands >200bp that do not code for proteins, are implicated in important processes of gene regulation, but little is known about their function in corals.

• Pocillopora sp. corals were experimentall enriched with dissolved inorganic nitrogen and phosphate for 15 months on an oligotrophic fore reef in Mo'orea, French Polynesia.
• Tissue samples were retrieved, went through RNA extraction, and were sent off for sequencing.
• The primary data type used in this study is RNA-seq data.

Objectives

1. Identify lncRNAs present within the RNA-seq data
2. Create a list of lncRNA transcript IDs
3. Examine the sensitivity of lncRNA expression to low level nutrient enrichment with differential expression analysis

Data

RNA-seq data for 16 control (no low level nutrient exposure) and 16 treatment (low level nutrient exposure) colonies.

• GitHub Repo
• RNA-seq data
• P. ver genome assembly (GFF3 and fasta)
• P. ver genome assembly GTF

Project Context

Original Citation: OSF

We experimentally enriched corals with dissolved inorganic nitrogen and phosphate for 15 months on an oligotrophic fore reef in Mo'orea, French Polynesia. Our physiological analysis of coral fragments and thermal performance show that in an oligotrophic fore reef environment, nutrient enrichment can cause changes in coral endosymbiont physiology that increase the performance of the coral holobiont. To assess the molecular underpinnings of the enhanced thermal performance due to chronic low nutrient enrichment, we assessed gene expression with RNASeq and the potential for gene expression regulation through whole genome bisulfite sequencing (WGBS). Sequencing of mtORF for 32 samples of Pocillopora spp. (n=16 per treatment) identified three possible species (Pocillopora meandrina, Pocillopora eydouxi, and an unidentified variant). To delineate between Pocillopora meandrina and Pocillopora eydouxi we will be using a new set of primers following methods found in Johnston et al. 2018.

Workflow for identifying lncRNAs in all 32 samples

Filtering Results

• List of lncRNA transcript IDs in GTF format

Differential Expression Analysis

• Count Matrix of lncRNAs

Projected Endpoint

Endpoint is getting pushed according to how quickly I am able to finish with enough time to prep the final project

• Original endpoint: Canonical list of lncRNAs for this experiment [complete]
• New endpoint: Differential expression analysis of lncRNAs (nutrient exposure vs control) with blast results of 22 significantly differentially expressed lncRNAs

Final Plan

• Week 6-7: Kallisto count matrix of lncRNAs
• Week 8-9: DESeq2 to examine lncRNA differential expression based on nurtrient treatment
• Week 10: figure generation, blast of lncRNAs, and final compendium

Weekly Log

Final week and updates

• Final zenodo release
• Finsished differential expression figures
• Finished compendium with full Rpub presentation in Quarto
• Cleaned up repo

Week 09

• Count matrix filter for only lncRNAs

• DESeq2 run

• 22 significantly differentially expressed lncRNAs out of ~6000

Week 08

• updates to count matrix code

• kallisto run to retrieve count matrix, filter needed

• worked on Rpub formatting and figure mock-up, view here

Week 07

• 07-scale-up code, with workflow up to original endpoint, fixed and cleaned up

• Tried using stringtie for count matrix, switching to kallisto

• R pub for final project

Progress Tracking

• Progress update presentation 04/27
• Project R pub in quarto 05/12