Transcriptional_Compensation

Data

Raw read files for RNA-seq of the Cadenza and Kronos EMS-mutagenised lines can be found in the ENA under project PRJEB89501.

The data from Xiong et al. (2020) is described in this paper: Transcriptome sequencing reveals hotspot mutation regions and dwarfing mechanisms in wheat mutants induced by γ-ray irradiation and EMS

Analysis of RNA-seq Data

The same pipeline was used for the Cadenza lines (scripts here), Kronos lines (scripts here), and the EMS-mutagenised lines from Xiong et al. (2020) (scripts here).

Preparing Reference Transcriptome (Kronos only)

• Filter Chinese Spring reference transcriptome to remove D-subgenome transcripts: filter_reference_fasta.sh, requires the Chinese Spring reference transcriptome available here
• Prepare kallisto index: kallisto_index.sh

Differential Expression Analysis

• Pseudoalignment and quantification with kallisto: kallisto.pl (requires the Chinese Spring reference transcriptome available here
• Import quantifications and summarise at the gene level: import_kallisto_quantifications.R
• Identify differentially expressed genes in the mutagenised lines: differential_expression_DESeq2_all_contrasts.R

Variant Calling

• Trim reads: trim.pl
• Map reads to Chinese Spring reference genome: hisat.pl, requires the Chinese Spring reference genome available here
• Call variants: call_variants_by_chrom.pl
• Predict variant effects: vep.sh
• Filter variants to keep only PTC variants or synonymous variants: vep_filter_PTC.sh or vep_filter_synonymous.sh
• Create lists of genes in diads or triads: genes_in_diads_triads.R
• Further filtering to keep variants that are homozygous mutant in at least one of the EMS-mutagenised lines, affect canonical transcripts, and are in a diad/triad: vep_output_filtering_PTC.R or vep_output_filtering_synonymous.R

Combine Differential Expression and Variant Calling

• Identify potential homoeologous transcriptional compensation associated with PTC or synonymous mutations and plot: up_down_regulated_homoeologues_PTC.R or up_down_regulated_homoeologues_synonymous.R