SCReadCounts Usage

Synopsis

Graphical User Interface:

scReadCounts

Command-line:

scReadCounts -r <bam_file> -s <snv_list_file> -o <output_file> [options]

Description

SCReadCounts has two programs. The program readCounts requires two input files: a pooled single cell alignment and a list of genomic positions of interest. readCounts utilizes the barcode information from the pooled single cell alignments and outputs the variant and reference read counts (n_var and n_ref, respectively), for each barcode (cell) present in the barcodes.tsv file, in a tab separated text file. This file is then used as an input for the second program - readCountsMatrix - which, upon providing an output prefix, generates two outputs: (1) a cell-position matrix with n_var and n_ref estimates, and (2) a cell-position matrix with the expressed variant allele fraction (VAF_RNA = n_var / (n_var + n_ref)). VAF_RNA is estimated at a user-defined threshold of minimum required sequencing reads (minR); default minR = 5. readCountsMatrix is time-efficient and can be re-run multiple times at various minR thresholds.

Some methods for extracting cell-barcodes from the BAM files (see Read Grouping) can use a file of cell-barcodes to restrict these to an acceptable list. The STARsolo cell-barcode read grouping method, for example, presumes valid cell-barcodes are in the file barcodes.tsv and the current working directory (from where the script is executed). See also, the Valid Read Groups options (-b, –barcode_acceptlist command-line options) below.

Graphical User Interface

Click the help icon (question mark) at the top right of the GUI and then an input field for help. Multiple files can be selected in the file-chooser using Ctrl-Click or Shift-Click. Fields can be reset to their default values using the Reset button. Click OK to execute SCReadCounts.

Additional GUI option tabs are documented below.

Options

SNVs, -s SNVS, –snvs=SNVS

Single-nucleotide-polymophisms (SNVs). Tabular and VCF format SNVs are supported. Multiple files are specified inside quotes, separated by spaces, and by using file globbing. The list of genomic positions of interest is accepted in a tab-separated format with no header, and contains the chromosome, position, reference and variant nucleotide. Examples of genomic positions of interest include single nucleotide variant (SNV) sites, somatic mutations, or RNA-editing loci. List of genomic positions of interest can be generated from a variant call on the corresponding datasets, or pre-defined from existing sources, such as COSMIC or dbSNP. See Input Files for more information.

Read Alignment Files, -r ALIGNMENTS, –readalignments=ALIGNMENTS

Read alignments files in indexed BAM format, with extension .bam. BAM index with extension .bam.bai must be located in the same directory. Multiple files are specified inside quotes, separated by spaces, and by using file globbing. scReadCounts accepts alignment files generated by popular aligning tools; the test dataset uses a STAR-generated alignment. See Input Files for more information.

Cell Barcode, -C CELLBARCODE, –cellbarcode=CELLBARCODE

Group reads based on cell-barcodes extracted from read name/identifiers or BAM-file tags. See Read Grouping for more details. Default: UMI-tools.

UMI Count, -U UMICOUNT, –umicount=UMICOUNT

Count unique identifiers (UMI) based on read name/identifiers or BAM-file tags. Default: None, count reads not UMIs.

Alignment Filter, -f FILTER, –alignmentfilter

Alignment filtering strategy. See Read Filtering for more details. Default: Basic.

Output Folder, -o OUTPUT, –output=OUTPUT

Output file. Requires extension-specific filenames. Accecptable extensions: csv, tsv, xls, xlsx eg: output.csv. Note: All the 3 outputs will have this extension.

–version

Show program’s version number and exit.

-h, –help

Show program help and exit.

Advanced Options

Min. Reads, -m MINREADS, –minreads=MINREADS

Minimum number of good reads at each SNV locus per alignment file. Default=5. This affects only VAF calculations.

Max. Reads, -m MAXREADS, –maxreads=MAXREADS

Scale read counts at high-coverage loci to ensure at most this many good reads at SNV locus per alignment file. Values greater than 1 indicate absolute read counts, otherwise the value indicates the coverage distribution percentile. Default=No maximum.

Directional, -D, –directional

Output directional (forward and reverse complement) VAF and read counts. Default: False

Valid Cell Barcodes, -b BARCODES, –barcode_acceptlist BARCODES

File of white-space separated, acceptable cell-barcodes. Overrides accept list, if any, specified by Cell Barcode option. Use None to remove a default accept list.

Threads, -t THREADS, –threads=THREADS

Worker threads. Default: 0, indicating single-threaded serial execution.

Force, -F, –force

Force all output files to be re-computed, even if already present. Default: False.

Quiet, -q, –quiet

Quiet. Default=verbose.