CLI Reference
usage: lbqc [-h] [-v] -i INPUT -o OUTPUT [-p PLASMID] -b BARCODES [-l INSERT_LENGTH] [-f FLANKS] [-r ENZYMES] [-a] [-T]
[-S] [-z ZSCORE] [-N EXPECTED_INSERTIONS] [--full-output]
Analyze long-read barcodes and generate a summary report
options:
-h, --help show this help message and exit
-v, --version Show the LongBarcodeQC version and exit.
-i INPUT, --input INPUT
Path to input fastq files (nanopore fastq_pass folder).
-o OUTPUT, --output OUTPUT
Path to output directory. An output folder will be created if it does not exist. Program will exit
if files already exist here.
-p PLASMID, --plasmid PLASMID
Path to plasmid fasta file. The default plasmid is AP-Amp. If using another plasmid, use this
option with the path to a fasta file containing your plasmid. For best results, concatenate the
circular plasmid sequence to itself to optimize alignment.
-b BARCODES, --barcodes BARCODES
Barcode FASTA file. Use a preset keyword or provide a path to a custom FASTA file. Preset options:
EV (Expression Vector), AP (Assembly Plasmid), TS (TritSeq). Custom file: path to a FASTA file with
one barcode per entry; headers must be unique.
-l INSERT_LENGTH, --insert_length INSERT_LENGTH
Integer length of expected insert size cloned into MCS. A rough estimate is sufficient. Default:
300 bp
-f FLANKS, --flanks FLANKS
Path to fasta file containing MCS flanking regions. The first sequence must be the upstream flank,
and the second sequence must be the downstream flank. Must be provided in the same file.
-r ENZYMES, --enzymes ENZYMES
Path to text file with restriction site names and their sequences. Define one site per line, with
the name and sequence separated by comma.
-a, --AP Option to add AP-Amp and AP-Kan alignment. Mainly used to detect contamination from assembly
plasmid reads. This will flag AP reads in the final output and score barcodes against them.
-T, --trim Trim the ONT Rapid (RAP) adapter and its leader sequence from reads before alignment, using
cutadapt. Off by default.
-S, --SBARRO Use this option if the plasmid is part of the SBARRO system (derived from c.18). Reference will be
generated with NNN sequence inserted into the MCS (length of NNNs equal to provided insert length).
-z ZSCORE, --zscore ZSCORE
Optionally set z-score threshold for barcode calling. The default threshold is set by using the
z-score for the worst performing barcode alignment in the library (absolute value). Sometimes this
value is either too conservative or too permissive. See the z-score distribution in the html output
summary to evaluate this. If desired - use this option to manually set the z-score threshold to be
a fixed value (e.g. 2.5).
-N EXPECTED_INSERTIONS, --expected_insertions EXPECTED_INSERTIONS
Optionally set the expected number of insertions in a library. This is used in the html report to
categorize reads in the read length histogram. By default, this is set to the maximum number of
sites/positions found in the dataset.
--full-output Write the full barcode alignment scores as a parquet file (includes MCS sequences and all per-
barcode scores). This file can be large.