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Configuration

IsoTV has two configuration files - environment.yaml, which handles the necessary packages and dependences for the conda environment, and config.yaml, which covers the general and specific pipeline parameters and paths. The overview to set up config.yaml is described below.

Pipeline Configuration

The pipeline configuration is split into three parts: general, processing, analysis, and tools. There are file paths for files and tools that need to be set before running. Specific module specific configuration is discussed in Modules.

The below configuration ids can be configured in the config.yaml file or explicitly specified in the command line.

General

outdir:/path/to/output - Path to output directory

basecalling: FALSE - Boolean to decide whether reads should be basecalled. preprocess: TRUE - Boolean to decide whether to use the generated files from processing for isoform analysis or not. Refers specifically to the nanopore_gtf, polished_reads, and counts_data configuration for the Analysis. annotation: TRUE - Boolean stating to use input annotation file. quantification: TRUE - Boolean stating to use input quantification (isoform expression) file.

ONT long read processing

guppy: "/path/to/Guppy324/bin/guppy_basecaller"
flowcell: FLO-MIN106
kit: SQK-DCS109
  • Path to Guppy basecaller, and the information regarding the ONT flowcell and kit number.
genome_fasta: "/path/to/GRCh38.p12.primary_assembly.genome.fa"
genome_annot: "/path/to/gencode.v32.primary_assembly.annotation.gtf"
  • Path to human genome primary assembly FASTA and GTF file. Recommendation is to use GRCh38.p12 and Gencode v32 respectively.
samples:
  A549_1: "A549_r1_r3"
  A549_2: "A549_r2_r1"
  ...
  • Mapping of sample fasta filename to desired name. Sample name with .fastq extension. For reference, the notation dayX_Y:"Z" corresponds of the X day and Y replicate from the fastq file Z of the experiment. This notation is required.

Feature Analysis

gene_file: "/path/to/genes.tab"
  • Input gene file to determine which genes to analyze. Genes are separated by a new line.
output_plots: "test.pdf"
  • Output report filename to store the visualizations from all the genes required.
nanopore_gtf: "/path/to/nanopore.gtf"
  • Path to gtf transcript file that was mapped to the de novo transcriptome. Required if not using NanoIso processing.
  • NOTE: The transcript ids should match with the transcript ids from the polished_reads and counts_data files.
polished_reads: "/path/to/corrected_transcriptome.fa"
  • Path to reads with polished sequences. Required if not using NanoIso processing.
  • NOTE: The transcript ids should match with the transcript ids from the nanopore_gtf and counts_data file.
counts_data: "/path/to/counts.txt"
  • Path to the normalized transcript counts data. Required if not using NanoIso processing.
  • NOTE: The transcript ids should match with the transcript ids from the nanopore_gtf and polished_reads file.
continuous: FALSE
  • Boolean to let pipeline know if data is continuous or not.

External tool paths

aa: TRUE
  • Boolean if amino acid sequence should be visualized.
iupred2a: TRUE
iupred2a: "/path/to/iupred2a/iupred2a.py"
  • Boolean if disorder regions should be predicted and visualized.
  • Path to IUPred2A python file script.
brewery: TRUE
brewery_path: "/path/to/Brewery/Brewery.py"
  • Boolean if secondary structure should be predicted and visualized.
  • Path to Porter5 python file script.
pfScan: TRUE
prositeScan_path: "/path/to/ps_scan/ps_scan.pl"
pfScan_path: "/path/to/ps_scan/pfscan"
prositeDat_path: "/path/to/prosite.dat"
  • Boolean if post-translational modifications should be predicted and visualized
  • Path to the Prosite scan perl file script and folder, and Prosite database.
java: "/pkg/openjdk-11.0.3.2-0/profile"
  • Path to java. Only necessary if local Java is not at least version 11.