CONFIGURATION_PARAMETERS.md

Custom Configuration Variables

Within each custom configuration file the following variables can be defined:

Table of Contents

1. Base Variables
2. STAR Variables
3. FeatureCounts Variables
4. Gene Extension Variables
5. Taxonomic-classification Variables
6. Filtering Variables
7. External Pipeline Variables

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Base Variables

Variable	Required/Optional	Description
input	Required	Path to the samplesheet.
outdir	Required	Path to the results/output directory; must exist before running.
bc_whitelist	Required	Path or link to the barcode whitelist file(s). If it's a link, it will be automatically downloaded and unzipped if applicable.
protocol	Required	Specifies the sequencing technology used (must be one of the following: "oak_seq", "10xv1", "10xv2", "10xv3", "10xv4", "parse_biosciences_WT_mini" or "parse_biosciences_WT", "bd_rhapsody", "sciRNAseq3" , "ultima_genomics" or "seqspec").
ref_fasta	Required	Path to the genome FASTA file used for mapping reads.
ref_gtf	Required	Path to the GTF/GFF file formatted for STARsolo.
ref_gtf_alt	Optional	Path to the GTF/GFF file formatted specifically for analysis with Parse Biosciences / CellRanger pipeline. Defaults to the same path as ref_gtf.
run_method	Optional	Method of running the pre-processing pipeline, demonstrated in the pipeline diagram, currently either "standard" or "geneext_only". Default is set to "standard".
mapping_software	Optional	Software used to map reads (must be one of the following: "starsolo", "alevin", "both"/"alevin_starsolo" or "alevin_subsampled_starsolo"). Default set to "starsolo".
perform_demultiplexing	Optional	Boolean flag to enable or disable demultiplexing of the FASTQ files, where applicable. Default is true.
seqspec_file	Optional	Path to the seqspec file.
subsample_nreads	Optional	The size (number of reads) of the subset used to map to STARsolo, in case the parameter mapping_software = alevin_subsampled_starsolo. Default set to 100000000 reads.

STAR Variables

Variable	Required/Optional	Description
star_index	Optional	Path to the pre-generated STAR index. By default the STAR index is created within the pipeline.
star_genomeSAindexNbases	Optional	Lenght of the SA pre-indexing string in STAR. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_genomeSAsparseD	Optional	Suffix array sparsity in STAR. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_solocellfilter	Optional	Cell filtering type and parameters used by STAR. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_soloFeatures	Optional	Genomic features for which the UMI counts per Cell Barcode are collected. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_outFilterScoreMin	Optional	Alignment will be output only if its score is higher than or equal to this value normalized by read length. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_outSAMunmapped	Optional	Output of unmapped reads in the SAM format. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_outSAMattributes	Optional	String of desired SAM attributes. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_generateBAM	Optional	Boolean to determine if a BAM file should be generated. Will automatically adjust the star_outSAMattributes. Default is set to true.
star_soloTypestring	Optional	String of defining soloType and barcode structure. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_soloCBmatchWLtype	Optional	Type of matching the cell barcodes to the barcode whiteList. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_soloUMIfiltering	Optional	Type of UMI filtering. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_soloMultiMappers	Optional	Counting method for reads mapping to multiple genes. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_soloUMIdedup	Optional	Type of UMI deduplication algorithm. See protocol-specific defaults set in the seqtech_paramaters.config file.
star_clipAdapterType	Optional	Type of adapter clipping. See protocol-specific defaults set in the seqtech_paramaters.config file.

FeatureCounts Variables

Variable	Required/Optional	Description
perform_featurecounts	Optional	Boolean flag to enable or disable calculation of mtDNA & rRNA percentages. Default is false.
mt_contig	Optional	Name of the mitochondrial contig in the reference annotation, used to calculate mtDNA content. Default set to "chrM M MT".
grep_rrna	Optional	String used to grep ribosomal RNA (rRNA) reads from annotations. Default set to "rRNA"

Gene Extension Variables

Variable	Required/Optional	Description
perform_geneext	Optional	Boolean flag to enable or disable the gene extension step after mapping. Default is false.

10x_saturate Variables

Variable	Required/Optional	Description
perform_10x_saturate	Optional	Boolean flag to enable or disable the 10x_saturate step after mapping. Default is true.
saturation_target	Optional	The saturation target fraction used to predict the input reads needed. Default set to 0.7.

Taxonomic-classification Variables

Variable	Required/Optional	Description
perform_kraken	Optional	Boolean flag to enable or disable Kraken2 classification of unmapped reads. Default is false.
kraken_db_path	Optional	Path to the Kraken2 database used for taxonomic classification of unmapped reads, if empty, a default database will be installed.

Filtering Variables

Variable	Required/Optional	Description
perform_cellbender	Optional	Boolean flag to enable or disable removal of ambient RNA using CellBender. Default is false.
cellbender_extraargs	Optional	Provide extra arguments to the CellBender function as a string. Refer to the CellBender manual for options.

External Pipeline Variables

Variable	Required/Optional	Description
perform_cellranger	Optional	Boolean flag to enable or disable the CellRanger pipeline. Default is false.
rhapsody_installation	Optional	Path to the BD-Rhapsody pipeline installation folder.
splitpipe_installation	Optional	Path to the split-pipe installation folder.
splitpipe_conda_env	Required (if splitpipe_installation is provided)	Path to the split-pipe conda environment created by following these instructions, required if running the split-pipe pipeline.
scalerna_installation	Optional	Path to the base of the ScaleRna pipeline installation folder.
scalerna_scalePlex	Required (if scalerna_installation is provided)	Boolean flag if the data is a ScalePlex dataset.
scalerna_libStructure	Required (if scalerna_installation is provided)	Scale scRNA assay version, should be one of the following: "libV1.json", "libV1.1.json", or "libQuantumV1.0.json"