Musketeer-D / WheatHomoeologExpression

WheatHomoeologExpression

Data

All the input data for the scripts described can be found here.

The data for the jupyter notebooks are here

Methods for Transcriptome paper.

This repository contains the code as used for the following sections

Homoeolog specific mapping by kallisto in nulli-tetrasomic lines

• plot heatmaps for nullitetra, all genes (not just triads)
• get expression for nullitetras 1:1:1 triads
• add dominance category to nullitetra 1:1:1 triad expression
• plot and stats for A vs B vs D for nullitetras and dominance categories

Genome of origin effect on gene expression

• plot clustering of intermediate tissue expression, colour by genome of origin

Expression complexity

Details in Jupyter notebook

Expressed genes within the Azhurnaya developmental time course (209 samples) and Chinese Spring no stress (123 samples).

Details in Jupyter notebook

Differential expression (Azhurnaya time course)

• differential expression all tissues compared to anther
• differential expression all tissues compared to awns
• differential expression all tissues compared to embryo
• differential expression all tissues compared to endosperm
• differential expression all tissues compared to flag leaf blade
• differential expression all tissues compared to flag leaf sheath
• differential expression all tissues compared to glumes
• differential expression all tissues compared to grain hard
• differential expression all tissues compared to grain milk
• differential expression all tissues compared to internode
• differential expression all tissues compared to leaf blades excl flag
• differential expression all tissues compared to leaf ligule
• differential expression all tissues compared to leaf sheaths excl flag
• differential expression all tissues compared to peduncle
• differential expression all tissues compared to root apical meristem
• differential expression all tissues compared to seedling aerial tissues
• differential expression all tissues compared to shoot apical meristem
• differential expression all tissues compared to shoot axis
• differential expression all tissues compared to spike
• differential expression all tissues compared to spikelets
• differential expression all tissues compared to stigma ovary
• after differential expression analysis filter differentially expressed genes for padj < 0.05
• after filtering padj < 0.05 summarise differentially expressed genes

eFP browser

The browser is available here

Relative expression levels of the A, B, and D genome homoeologs across triads

Details in Jupyter notebook

Definition of homoeolog expression bias categories

Details in Jupyter notebook

Analysis of effect on polyploidy on homoeolog expression bias

Details in Jupyter notebook

Variation in homoeolog expression bias across tissues (Stable/Dynamic triads).

Details in Jupyter notebook

CDS, protein and promoter analysis for triads

The scripts used for this section are in various repositories:

• Extract promoter regions
• Pairwise alignment of triads

Transposable Element (TE) modeling using CLARITE

Clarite is available here

TE presence in gene promoters

• Calculate sliding window of Transposable Element density upstream of genes
• Calculate average lengths and distances from start site of Transposable Elements including statistical tests
• Script for analysis of TE presence/absence based on homoeolog expression bias categories.
• Script for analysis of TE presence/absence based on homoeolog expression bias variation categories.

Enrichment of TE families in gene promoters

• Script for analysis of TE family enrichment based on homoeolog expression bias variation categories.

TE density in gene promoters

• Plot sliding window of Transposable Element density including statistical tests

Transcription factor binding site (TFBS) identification

• Analyse TFBS categories according to triad movement including statistical tests
• Filter unique FIMO hits only - step 1
• Generate TFBS categories based on occurrence in triads
• Filter unique FIMO hits only - step 2
• Run FIMO to identify TFBS in promoters

Ka/Ks analysis

• [MAFFT alignment](https://github.com/TGAC/bioruby-polyploid- tools/blob/master/bin/mafft_triads.rb.)
• Script to obtain Chinese Spring No-Stress analysis of Ka/Ks ratios for syntenic 1:1:1 triads.
• Script to obtain Azhurnaya development analysis of Ka/Ks ratios for syntenic 1:1:1 triads.
• Script to obtain Chinese Spring No-Stress analysis of Ka/Ks ratios for non-syntenic 1:1:1 triads.
• Script to obtain Azhurnaya development analysis of Ka/Ks ratios for non-syntenic 1:1:1 triads.

WGCNA Network construction

• WGCNA combine studies filter normalise samples for abiotic.R
• WGCNA combine studies filter normalise samples for disease.R
• WGCNA combine studies filter normalise samples for grain.R
• WGCNA combine studies filter normalise samples for leaf.R
• WGCNA combine studies filter normalise samples for root.R
• WGCNA combine studies filter normalise samples for spike.R
• WGCNA determine threshold for abiotic.R
• WGCNA determine threshold for disease.R
• WGCNA determine threshold for grain.R
• WGCNA determine threshold for leaf.R
• WGCNA determine threshold for root.R
• WGCNA determine threshold for spike.R
• WGCNA run WGCNA abiotic.R
• WGCNA run WGCNA disease.R
• WGCNA run WGCNA grain.R
• WGCNA run WGCNA leaf.R
• WGCNA run WGCNA root.R
• WGCNA run WGCNA spike.R

Defining same, similar and divergent expression patterns of triads

• calculate the distance between homoeologs
• plot graphs of distances between homoeologs

Module overlaps

• compares whether modules in different tissue networks have significant overlap in terms of genes in each module

Gene tree for MADS_II in root module 61

• blast MADSII proteins against other wheat proteins

Correlation to stress status

• calculate stress correlations for abiotic
• calculate stress correlations for disease

Genie3

• Results from Genie3

Identifying high connected hub genes

• find hub genes in abiotic network
• find hub genes in disease network