Introduction

The idea for this project came out of recent research the RNA editing capabilities housed within the genome of cephalopods.
Found that RNA (instead of DNA) is highly edited in Octopus sp.
- Occurs mostly in neurological tissue, but can also occur in other peripheral tissues.
Cuttlefish have not been studied.
In preparing for RNA transcriptome analysis, we realized that we did not have a reference sequence to compare our transcriptome analysis to, so we decided to sequence the genome as well.

Sepia bandensis

Mollusca (phylum) —> Cephalopoda (class)
Found off the coast of Philippines, Papua New Guinea and Indonesia in shallow water (water of 78-80F)
Babies are about 0.5in, adults are up to 2-3 inches.
8 arms, with two rows of suckers along each arm, and two feeding tentacles with at least two rows of suckers along each
Cephalopods have three hearts, a ring shaped brain, blue, copper based blood, and have a lifespan between 6 months and 3 years (Sepia bandensis lives about a year)
Chromatophores and skin shaping used to hide from predators/hunt
The cephalopod genome is roughly 2.7GB.
- There is no completed sequencing data on Cuttlefish (S. officianalis is being analyzed by Kim Worley’s group out of Baylor).
- We do not know the size of S. bandensis’ genome.
- We also don’t know how many chromosomes the organism has. S. pharaonic has 48 chromosomes (2N)
A recent paper showed that RNA editing occurs at greater levels in colder water. Will be interesting to highlight RNA editing in warm water fish

A biological process in which an organism increases its adaptability by editing mRNA transcripts
Can be paired with gene splicing (i.e. in humans)
A to I RNA editing occurs when the adenosine is edited to an Inosine via deamination.
- The resultant nucleotide substitution is a Guanine/Guanosine

RNA editing allows for the expression of functionally diverse proteins from a single gene (i.e. multiple transcripts from one gene) = enhanced adaptability
- I.e. in an environment where the surrounding temperature changes it is useful to have functional variants of a protein that allows for an increase in ion permeability through the membrane (i.e. increased action potential)
Studying RNA editing in an organism allows us to answer questions about how the organism adapts to its environment.

Karyotype the organism (how many chromosomes does it have).
Perform NGS of S. bandensis gDNA.
- Annotate the genome of S. bandensis (using S. officianalis genomic data as a scaffolding reference).
Perform RNA Transcriptome analysis of all tissues.
- Assess frequency of A to G substitutions within the transcriptome.
- Determine which genes are edited

Cuttle Wranglin’

Setting up a salt water reef tank
Maintaining the fish until adulthood Tissue Processing
Karyotyping
Isolating neurological tissue and peripheral tissue
Prepping gDNA, sending frozen tissues off for RNA library production Data analysis
Genome assembly
Transcriptome analysis