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https://github.com/lotharukpongjs/hourglass_brownalga

Exploring the transcriptomic hourglass in three brown algal species.
https://github.com/lotharukpongjs/hourglass_brownalga

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Exploring the transcriptomic hourglass in three brown algal species.

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README 

        
# Transcriptomic hourglass in brown algae

Exploring the transcriptome evolution in three brown algal species (**_Fucus distichus_**, **_Fucus serratus_**, & **_Ectocarpus_ species 7**) across the entire life cycle. BioProject number (`PRJNA1090323`). In addition, analysis of two brown algal species with haplodiplontic life cycles (_Laminaria digitata_ and _Saccorhiza polyschides_) for taxonomic breadth.



## Documentation

I have rendered all the analysis (in Rmd) as HTML files. I have included all the necessary R scripts to generate the figures and interpretation of the brown algal developmental transcriptome datasets. Both `2_evolutionary_transcriptomics.Rmd` and `3_further_evolutionary_transcriptomics.Rmd` scripts can be run from the data available in the github repository (https://github.com/LotharukpongJS/hourglass_brownalga), which was generated using step 1 "Obtaining RNA-seq data and gene age data".

`4_address_reviewer_comments.Rmd` can also be run from the data available in the github repository.



### 1 – Obtaining RNA-seq data and gene age data (available [here](https://lotharukpongjs.github.io/hourglass_brownalga/1_preprocessing.html))

* RNA-seq quantification

* Gene age inference

* Processing RNA-seq and gene age data 

* Create input for myTAI

* Visualise gene age distribution

* Orthogroup abundance



### 2 – Evolutionary transcriptomics analyses (available [here](https://lotharukpongjs.github.io/hourglass_brownalga/2_evolutionary_transcriptomics.html))

* Transcriptome evolution in _Fucus_ embryogenesis

* Transcriptome evolution in _Ectocarpus_

* Transcriptome evolution across _Fucus_ tissues

* Mean expression of different PS



### 3 – Further evolutionary transcriptomics analyses (available [here](https://lotharukpongjs.github.io/hourglass_brownalga/3_further_evolutionary_transcriptomics.html))

* Transcriptome evolution in Fucus embryogenesis using RNA-seq that has been ‘denoised’ using noisyR

* Stats for male-female difference in Ectocarpus

* Tau profiles

* TDI across _Ectocarpus_ life cycle stages

* Visualisation of orthogroups

* pTAI analysis

* GO analysis of top contributor gene (from `pTAI`)



### 4 – Further evolutionary transcriptomics analyses (addressing reviewers) (available [here](https://lotharukpongjs.github.io/hourglass_brownalga/4_address_reviewer_comments.html)). 

NOTE: the version of myTAI is newer due to an addition of the PairwiseTest(). However, the lower-level functions such as `cpp_TAI` remain the same, thus, results from other functions should not differ.



* Difference in TAI between sexes and tissues (matSP reptip vs vegtip; matSP reptip vs all; matSP reptip male vs female; matSP gamete male vs female).

* Difference in TAI between multicell and unicell in _Ectocarpus_, as well as the reductive hourglass test in GA and PSP.

* Additional analysis of two *complex* brown algal species with haplodiplontic life cycles (_Laminaria digitata_ and _Saccorhiza polyschides_).

* Similarity/distance between _Ectocarpus_ and the embryo stages in the two species of _Fucus_.



## Version control



At the end of each of these scripts, I list the software and versions used. R code here was run on MacBook Pro (Apple M1 Chip, 16 GB Memory, macOS Ventura v13.4.1). Some of the preprocessing step (e.g., RNA-seq quantification, gene age inference, interproscan) were run on a high performance compute cluster. Otherwise, the rest of the script should take less than 30 minutes to run on a "normal" desktop computer.



Here is a summary of bioinformatic software used outside of base R (v4.2.2) packages and basic unix tools that were crucial to the analyses presented in this work. As mentioned previously, software versions are available at the end of each analysis script.



Selected software



- `nf-core/rnaseq` r3.5 https://nf-co.re/rnaseq/3.5

- `GenEra` v1.0 https://github.com/josuebarrera/GenEra

- `diamond` v2.0.14 https://github.com/bbuchfink/diamond

- `tidyverse` v2.0.0 https://www.tidyverse.org/

- `myTAI` v1.0.1.9000 https://github.com/drostlab/myTAI

- `OrthoFinder` v2.5.4 https://github.com/davidemms/OrthoFinder

- `orthologr` v0.4.2 https://github.com/drostlab/orthologr/

- `salmon` v1.5.2 https://github.com/COMBINE-lab/salmon

- `noisyR` v1.0.0 https://github.com/Core-Bioinformatics/noisyR

- `philentropy` v0.7.0 https://github.com/drostlab/philentropy

- `DESeq2` v1.36.0 https://github.com/thelovelab/DESeq2

- `interproscan` v5.61-93.0 https://github.com/ebi-pf-team/interproscan

- `topGO` v2.48.0 https://bioconductor.org/packages/release/bioc/html/topGO.html