/Native-Invasive_transcriptional_plasticity

Repository for scripts and workflow notes for project investigating transcriptional plasticity in native vs invasive fishes

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Native-Invasive transcriptional plasticity

Repository for scripts and workflow notes for project investigating transcriptional plasticity comparing two native and invasive fishes

Bioinformatics Analyses General Workflow

(sections align with subfolders in repository)

1. RNA-Seq Initial QC and Processing to counts

  • De-multiplexed and combined (concatenated) sequences across lanes for each sample
    • NGS.nested.for.loop.ahi.sh
  • FASTQC to assess initial data quality
  • Raw reads per sample filtered via adaptor trimming with Scythe and adaptive quality trimming with Sickle
    • sickleLK.sh & scytheLK.sh
  • Trimmed sequences aligned to previously generated reference transcriptomes for each species using BWA
    • BWA.mem.DS.ONLY-LK.sh, BWA.mem.Mb.ONLY-LK.sh,BWA.mem.singletons.DS.ONLY-LK.sh, BWA.mem.singletons.Mb.ONLY-LK.sh
  • Additional filtering to remove secondary and supplementary alignments to avoid issues of paralog and chimeric sequences.
    • samtools_filter_rm2reads.sh
  • Generate mapping stats
    • samtools_flagstats.sh, flagstat_tabbed.sh
  • Resulting filtered alignments processed to counts per transcript contig using SAMtools
    • bam_index_all.sh, samtools_idxstats.sh
  • Transdecoder to predit the translated peptide sequences within each transcriptome N.B. detailed HackMD document has additional info of output etc
    • transdecoder_ORF_DS.sh, transdecoder_ORF_Mb.sh, transdecoder_predict_DS.sh, transdecoder_predict_Mb.sh
  • Identified orthologues between the two transcriptomes using Orthofinder
    • Orthofinder.sh
  • Linked orthogroup output with the transcript count files for each species (filtered/rm2reads versions), roll up to orthogroup counts, combine to generate one combined count matrix for DGE (including all samples for both species, transcripts summed within the same orthogroup)
    • Orthologue_assignment.R

2. Differential Expression Analyses

  • Differential expression analyses using the limma-voom transformation and a general linear model
    • Limma_orthogroup_GH.R
  • Created graphs of LFC distribution shifts, DE reaction norms and heatmap for MS
    • Sig_genes_heatmaps_orthogroups_GHcp.R, Reaction_normgraph_GHcp.R
  • UpsetR to visualize shared and unique DE orthogroups between treatments
    • UpsetR_siggenes_shared_GHcp.R

3. Functional Analyses

  • Pre-processing: combined and removed duplicate GO terms for annotated transcripts within orthogroups
  • Assessed functional enrichment for a priori contrasts using Fisher’s exact tests as implemented in TopGO
    • TopGO_Orthogroup_Ghcp.R
  • Created similarity matrix using GoSemSim followed by hierarchical clustering for visualization of enriched biological processes, adapted from script and suggestions from Kyle Wellband, labels from Revigo and other database sources (manual investigation for each subgroup, finalized list of appropriate labels in excel: GO_groupings_0.92clustering_orthologsLMK). N.B. HackMD document has additional info of output, resources for decisions, etc
    • Enriched_GO_Fisher_orthogroup_comparisons&analyses_Ghcp.R, Species_orthogroup_compare_GOheatmap_BP_Ghcp.R