Simplex Basecalling
Quick start
To run Dorado basecalling, using the automatically downloaded hac model
on a directory of POD5 files or a single POD5 file use:
To basecall a single file, simply replace the directory pod5s/ with a path to your data.
To automatically download and use the fast or sup models try the following:
If you have a model that has already been downloaded you can specify that simplex model using a path. For more information on how models are downloaded and how they can be re-used please see the downloader documentation.
Adding modified bases
To add modified basecalling extend the model complex or refer to modified basecalling usage guide for more details on the other options available.
Selecting data
To basecall all reads in a nested directory structure recursively
use -r / --recursive:
To basecall only a limited number reads use the -n / --max-reads argument:
Tip
You can generate a list of read ids using the pod5 view tool.
To basecall a specific selection of reads use the -l / --read-ids argument passing in a file path
to a newline-delimited list of read ids. Only these read ids will be basecalled.
Resume basecalling
If basecalling is interrupted, it is possible to resume basecalling from a BAM file.
To do so, use the --resume-from flag to specify the path to the incomplete BAM file.
Warning
Do not reuse the filenames for --resume-from and the new output.
If they are the same then the interrupted file will be deleted when Dorado is launched and the previous work will be lost.
Read trimming
See read trimming.
Output Folder Structure
If the --output-dir <DIR> argument is set, Dorado basecaller will write output files
into a nested folder structure following the
MinKnow output structure specifications.
The chosen directory <DIR> becomes the root of the nested folder structure and replaces
/data/ in the specification examples.
Reads with mean Q-score below the --min-qscore threshold are written to the files marked fail.
If --min-qscore is not set, a default threshold of 0 is used and all reads are written to files marked pass.
CLI reference
Here's a slightly re-formatted output from the Dorado basecaller subcommand for reference.
Info
Please check the --help output of your own installation of Dorado as this page may be outdated
and argument defaults have been omitted as they are platform specific.
> dorado basecaller --help
Positional arguments:
model Model selection {fast,hac,sup}@v{version} for automatic model selection
including modified bases, or path to existing model directory.
data The data directory or file (POD5/FAST5 format).
Optional arguments:
-h, --help shows help message and exits
-v, --verbose [may be repeated]
-x, --device Specify CPU or GPU device: 'auto', 'cpu', 'cuda:all' or 'cuda:<id>[,<id>...]'.
Specifying 'auto' will choose either 'cpu', 'metal' or 'cuda:all' depending
on the presence of a GPU device.
--models-directory Optional directory to search for existing models or download new models into.
--bed-file Optional bed-file. If specified, overlaps between the alignments and
bed-file entries will be counted, and recorded in BAM output using
the 'bh' read tag.
Input data arguments:
-r, --recursive Recursively scan through directories to load FAST5 and POD5 files.
-l, --read-ids A file with a newline-delimited list of reads to basecall. If not provided,
all reads will be basecalled.
-n, --max-reads Limit the number of reads to be basecalled.
--resume-from Resume basecalling from the given HTS file. Fully written read records are
not processed again.
--disable-read-splitting Disable read splitting
Output arguments:
--min-qscore Discard reads with mean Q-score below this threshold or write them to
output files marked `fail` if `--output-dir` is set.
--emit-moves Write the move table to the 'mv' tag.
--emit-fastq Output in fastq format.
--emit-sam Output in SAM format.
-o, --output-dir Optional output folder which becomes the root of the nested output folder structure.
Alignment arguments:
--reference Path to reference for alignment.
--mm2-opts Optional minimap2 options string. For multiple arguments surround with
double quotes.
Modified model arguments:
--modified-bases A space separated list of modified base codes. Choose from:
pseU, 5mCG_5hmCG, 5mC, 6mA, 5mCG, m6A_DRACH, m6A, 5mC_5hmC, 4mC_5mC.
--modified-bases-models A comma separated list of modified base model paths.
--modified-bases-threshold The minimum predicted methylation probability for a modified base
to be emitted in an all-context model, [0, 1].
Barcoding arguments:
--kit-name Enable barcoding with the provided kit name. Choose from:
EXP-NBD103 EXP-NBD104 EXP-NBD114 EXP-NBD114-24 EXP-NBD196 EXP-PBC001
EXP-PBC096 SQK-16S024 SQK-16S114-24 SQK-LWB001 SQK-MLK111-96-XL
SQK-MLK114-96-XL SQK-NBD111-24 SQK-NBD111-96 SQK-NBD114-24 SQK-NBD114-96
SQK-PBK004 SQK-PCB109 SQK-PCB110 SQK-PCB111-24 SQK-PCB114-24 SQK-RAB201
SQK-RAB204 SQK-RBK001 SQK-RBK004 SQK-RBK110-96 SQK-RBK111-24 SQK-RBK111-96
SQK-RBK114-24 SQK-RBK114-96 SQK-RLB001 SQK-RPB004 SQK-RPB114-24
TWIST-16-UDI TWIST-96A-UDI VSK-PTC001 VSK-VMK001 VSK-VMK004 VSK-VPS001.
--sample-sheet Path to the sample sheet to use.
--barcode-both-ends Require both ends of a read to be barcoded for a double ended barcode.
--barcode-arrangement Path to file with custom barcode arrangement.
--barcode-sequences Path to file with custom barcode sequences.
--primer-sequences Path to file with custom primer sequences.
Trimming arguments:
--no-trim Skip trimming of barcodes, adapters, and primers.
If option is not chosen, trimming of all three is enabled.
--trim Specify what to trim. Options are 'none', 'all', 'adapters', and 'primers'.
Default behaviour is to trim all detected adapters, primers, or barcodes.
Choose 'adapters' to just trim adapters.
The 'primers' choice will trim adapters and primers, but not barcodes.
The 'none' choice is equivalent to using --no-trim.
Note that this only applies to DNA. RNA adapters are always trimmed.
Poly(A) arguments:
--estimate-poly-a Estimate poly(A/T) tail lengths (beta feature).
Primarily meant for cDNA and dRNA use cases.
--poly-a-config Configuration file for poly(A) estimation to change default behaviours
Advanced arguments:
-b, --batchsize The number of chunks in a batch. If 0 an optimal batchsize will be selected.