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plant-food-research-open/genepal: Output

Introduction

This document describes the output produced by the pipeline.

The directories listed below will be created in the results directory after the pipeline has finished. All paths are relative to the top-level results directory.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

Repeat annotation

Output files
  • repeatmodeler/
    • *.fa: Repeat library
  • edta/
    • *.EDTA.TElib.fa: Repeat library

A repeat library is created with either REPEATMODELER or EDTA. The choice of the tool is specified by the repeat_annotator parameter (default: repeatmodeler). Repeat annotation outputs are saved to the output directory only if save_annotated_te_lib parameter is set to true (default: false).

Repeat masking

Output files
  • repeatmasker/
    • *.masked: Masked assembly

Soft masking of the repeats is performed with REPEATMASKER using the repeat library prepared in the previous step. Masking outputs are saved to the output directory only if repeatmasker_save_outputs parameter is set to true (default: false).

RNASeq trimming, filtering and QC

Output files
  • fastqc_raw/
    • *.html: HTML QC report for a sample before trimming
    • *.zip: Zipped QC files for a sample before trimming
  • fastqc_trim/
    • *.html: HTML QC report for a sample after trimming
    • *.zip: Zipped QC files for a sample after trimming
  • fastp/
    • html/
      • *.fastp.html: HTML trimming report for a sample
    • json/
      • *.fastp.json: Trimming statistics for a sample
    • log/
      • *.fastp.log: Trimming log for a sample
    • *_{1,2}.fail.fastq.gz: Reads which failed trimming
    • *.paired.fail.fastq.gz: Pairs of reads which failed trimming
    • *.merged.fastq.gz: Reads which passed trimming. For paired reads, reads 1 and 2 are merged into a single file
  • sortmerna/
    • *.sortmerna.log: Filtering log for a sample
    • *_{1,2}.non_rRNA.fastq.gz: Filtered reads

RNASeq reads are trimmed with FASTP and are QC'ed with FASTQC. Ribosomal reads are filtered out using SORTMERNA. Trimmed reads are only stored to the output directory if the save_trimmed parameter is set to true (default: false). Reads filtered by SORTMERNA are stored to the output directory if the save_non_ribo_reads parameter is set to true (default: false).

RNASeq alignment

Output files
  • star/
    • alignment/
      • X.on.Y.Aligned.sortedByCoord.out.bam: Sorted BAM file of read alignments for sample X against reference Y
      • X.on.Y.Log.final.out: STAR final log file for sample X against reference Y
    • cat_bam/
      • Y.bam: A single BAM file for reference Y created by concatenating alignments from sample-wise *.on.Y.Aligned.sortedByCoord.out.bam files

RNASeq alignment is performed with STAR. Alignment files are only stored to the output directory if the star_save_outputs parameter is set to true (default: false). Concatenated bam files are stored to the output directory if the save_cat_bam parameter is set to true (default: false).

Annotation with BRAKER

Output files
  • etc/braker/
    • Y/
      • braker.gff3: Gene models predicted by BRAKER in GFF3 format
      • braker.gtf: Gene models predicted by BRAKER in GTF format
      • braker.codingseq: Coding sequences for the predicted genes
      • braker.aa: Protein sequences for the predicted genes
      • braker.log: BRAKER log file
      • hintsfile.gff: Evidential hints used by BRAKER in GFF format
      • what-to-cite.txt: A list of references which must be cited when reporting outputs created by BRAKER

BRAKER is used to annotate each genome assembly using the provide protein and RNASeq evidence. Outputs from BRAKER are stored to the output directory if the braker_save_outputs parameter is set to true (default: false).

Caution

BRAKER outputs are not the final outputs from the pipeline and that's why they are not stored by default. These are only intermediary files.

The pipeline further processes the BRAKER predictions and stores the final validated outputs in the annotations directory. The braker_save_outputs option is only provided to allow a manual resume of the pipeline for advanced use cases. See Advanced inputs for manual resume in the usage doc.

Annotation with Liftoff

Gene models are lifted from reference assembly(ies) to the target assembly using LIFTOFF. Currently, the outputs from Liftoff are considered intermediary and an option to store them in the output directory is not available.

Annotation filtering and merging

Annotations obtained from BRAKER and LIFTOFF are filtered with TSEBRA and merged with AGAT. Currently, the outputs from these processes are considered intermediary and an option to store them in the output directory is not available.

Functional annotation

Output files
  • annotations/
    • Y/
      • Y.emapper.annotations: TSV with the annotation results
      • Y.emapper.hits: TSV with the search results
      • Y.emapper.seed_orthologs: TSV with the results from parsing the hits, linking queries with seed orthologs

Functional annotation of the gene models from BRAKER and Liftoff is performed with EGGNOG-MAPPER.

Orthology inference

Output files
  • orthofinder/
    • Comparative_Genomics_Statistics/
    • Gene_Duplication_Events/
    • Orthogroups/
    • Phylogenetic_Hierarchical_Orthogroups/
    • Species_Tree/

If more than one genome is included in the pipeline, ORTHOFINDER is used to perform an orthology inference.

Final annotation files

Output files
  • annotations/
    • Y/
      • Y.gt.gff3: Final annotation file for genome Y which contains gene models and their functional annotations
      • Y.pep.fasta: Protein sequences for the gene models
      • 'Y.cdna.fasta': cDNA sequences for the gene models
      • 'Y.cds.fasta': Coding sequences for the gene models

The final annotation files are saved in GFF3 format validated with GENOMETOOLS and FASTA format obtained with GFFREAD.

Annotation QC

Output files
  • busco/
    • gff/
      • short_summary.specific.Y.eudicots_odb10.txt: BUSCO summary for annotations from genome Y against the eudicots_odb10 database
      • busco_figure: BUSCO summary figure including statistics for annotations from all the genomes
    • fasta/
      • short_summary.specific.Y.eudicots_odb10.txt: BUSCO summary for genome Y against the eudicots_odb10 database
      • busco_figure: BUSCO summary figure including statistics for all the genomes
  • etc/
    • splicing_marked/
      • Y.gff3: Final annotation file for genome Y which contains gene models and their functional annotations. Additionally, the intron features are marked as canonical or non-canonical and the splice motif is also added an attribute.

The completeness of the annotations is checked with BUSCO. To provide a comparative baseline, the completeness of the genomes is also checked. Moreover, the canonical/non-canonical splicing of the introns is also assessed by the pipeline.

Reports

Output files
  • multiqc_report.html: A MultiQC report which includes QC statistics, software versions and references
  • genepal_report.html: A specialized pangene analysis report
  • genepal_data/: Files containing parsed data from the reporting module

MultiQC and R Markdown are used to curate the results of the pipeline in two HTML reports. The MultiQC report is meant to serve as an exhaustive report of the pipeline outputs. Whereas, the R Markdown report (genepal_report.html) provides an overall summary along with a pangene analysis if multiple genomes are provided as input.

Pipeline information

Output files
  • pipeline_info/
    • Reports generated by Nextflow: execution_report.html, execution_timeline.html, execution_trace.txt and pipeline_dag.dot/pipeline_dag.svg.
    • Reports generated by the pipeline: pipeline_report.html, pipeline_report.txt and genepal_software_mqc_versions.yml. The pipeline_report* files will only be present if the --email / --email_on_fail parameter's are used when running the pipeline.
    • Parameters used by the pipeline run: params.json.

Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to troubleshoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage. MultiQC compiles a HTML report from the tools used by the pipeline.