Overview and Motivation:

Genetic variation 6 Acropora tenuis larvae from the Bioproject PRJDB6484. Following the CBC-Uconn variant calling tutorial. The motivation of this project is to attempt to understand genetic components of the Acropora tenuis genome. Genetic variation studies can identify highly conserved genes or genes with high amounts of variation. It can also identify structural variation such as single nucleotide polymorphisms, inversions, insertions, duplications, deletions, copy number variations, and more. Hopefully by understanding the genetic variation of this coral, we can understand and explain some more biological functions of the coral.

Structure:

1. Pre-processing
2. Variant Calling with bcftools and Freebayes
3. Filtering and comparing variants
4. Variant Annotation

Data:

We are downloading and using the 6 Acropora tenuis larvae genotypes from the Bioproject mentioned above, PRJDB6484. We will also be using the Aten_1.0 reference genome.

Our final variant called vcf files exist at /home/FCAM/EEB5300/usr6/DATA . This folder contains 3 subfolders; one for bcftools, one for freebayes, and one for the filtered dataset. Our working directory (with all error logs, raw data, sam and bam files, and everything inbetween) exists at /home/FCAM/EEB5300/usr6/Variant-Calling. The scripts used in our project were uploaded to the scripts folder as linked here.

Initial Questions: Our initial questiona were about heat stress and coral survival and what these particular warm water coral might have in the way of beneficial adaptations to avoid heat stress in todays's era of climate change and warming oceans. One notible study was done with A.Tenuis and looked at the heat resistance of different naturally occuring color morphs and they noted that the color variation of the coral itself did seem to lend a heat resistance to the coral. The color morph most likely to be heat resistant were those corals that were green in color, next were those with a purple color morph and final the least heat resistant were the brown color morphs which are considered the wild type for this species of coral (Noriyuki et al 2018). We had also hoped to find variation in genes related to the structural function and growth of these corals.

Discussion: We originally thought that this project would be a good example of variant calling in a non model species of coral, howevere the issue with using a non model species is that sometimes( I would venture frequently) things do not go to plan, that was certaintly the case. We did our best to research each new problem that arose and find solutions. However since the initial issues mentioned in our pre- processing segment took so much of our time to run down what was going wrong, and then we had issues with our second vaariant calling software mentioned in the Variant calling segment, by the time we got to the issues of anotating our data and our attempts to build a data base to compare the GFF file and our variants we realized that might have to sart all over again with a different reference genome or try to find information on how to align the GFF file and our reference genome we just plain ran out of patience and time. Overall though this project has been a very edicational experience and we have learned quite alot about variant calling and bioinformatics in general and if we attempted to do a project like this again we now know how to avoid the first few groups of errors we had and have learned a great deal about all of the programs that we have used on this project.

Required software:

• Quality control: FASTQC, sickle
• Alignment: bwa, samtools, picard, samblaster, bamtools, igv
• Variant Calling: bcftools, freebayes
• Other: bedtools, tabix, bgzip

Related Work/Citations:

• https://www.ncbi.nlm.nih.gov/bioproject/PRJDB6484
• https://www.ncbi.nlm.nih.gov/assembly/GCA_014633955.1/
• https://ftp.ncbi.nih.gov/genomes/all/GCA/014/633/955/GCA_014633955.1_Aten_1.0/
• https://github.com/CBC-UCONN/Variant-Calling
• https://www.biorxiv.org/content/10.1101/2020.08.28.272823v1.full
• https://academic.oup.com/g3journal/article/11/2/jkab018/6114454
• http://www.coralsoftheworld.org/species_factsheets/species_factsheet_summary/acropora-tenuis/
• https://reefbuilders.com/2021/01/09/the-bali-aquarium-armageddon-tenuis-is-just-mind-blowing/
• https://oceanservice.noaa.gov/education/tutorial_corals/coral07_importance.html
• https://marinegenomics.oist.jp/aten/viewer/download?project_id=97