Read Trimming
Dorado can trim adapters and/or primer sequences from the beginning and end of DNA and RNA reads during basecalling
For DNA basecalls only, trimming can be done as a separate step after basecalling
using the Dorado trim subcommand.
RNA trimming
RNA trimming is always done in-line with basecalling and cannot be done afterwards
using Dorado trim.
Demultiplexing trimmed data
Trimming adapters and primers may result in parts of the barcode flanking regions being removed, which could interfere with demultiplexing.
Trimming while basecalling
Dorado basecaller will attempt to detect any adapter or primer sequences at
the beginning and end of reads, and remove them from the output sequence. The
sequences searched for will depend on the sequencing-kit used, which is normally
embedded as metadata within pod5 files. Note that by default only kit14 sequencing
kits are supported, so if an older or non-standard kit was used, no adapter or
primer trimming will be performed.
Dorado will also attempt to infer the orientation of the read from any detected primers.
If the orientation can be inferred, then the output BAM record for the read will include
the TS:A:[+/-] tag, with a + indicating 5' to 3' orientation, and a - indicating
3' to 5' orientation.
In the specific cases of the SQK-PCS114 and SQK-PCB114 sequencing kits, if a UMI tag is
present, it will also be detected and trimmed. Additionally, the UMI tag, if found, will
be included in the BAM output for the read using the RX:Z tag.
This functionality can be controlled using either the --trim or --no-trim options
with Dorado basecaller. Note that if primer trimming is not enabled, then no attempt
will be made to detect primers, or to classify the orientation of the strand based on
them, or to detect UMI tags.
The --trim option takes as its argument one of the following values:
| Option | Adapters | Primers | Barcodes | Description |
|---|---|---|---|---|
all |
Detected adapters or primers will be trimmed. If barcoding is enabled, detected barcodes will be trimmed. This is the default option |
|||
adapters |
Detected adapters will be being trimmed, but primers will not be trimmed. If barcoding is enabled, detected barcodes will not be trimmed. |
|||
none |
Nothing will be trimmed. Equivalent to --no-trim |
Trimming existing datasets
The Dorado trim subcommand can be used to trim adapters and/or primer sequences in
existing basecalled datasets. To do this, run:
<calls> can either be an HTS format file (e.g. FASTQ, BAM, etc.) or a stream of an
HTS format (e.g. the output of Dorado basecalling).
<kit> must be provided to specify the sequencing kit used, since this is not encoded in FASTQ or BAM files.
The --no-trim-primers option can be used to prevent the trimming of primer sequences.
In this case only adapter sequences will be trimmed.
If it is also your intention to demultiplex the data, then it is recommended that you demultiplex before trimming any adapters and primers, as trimming adapters and primers first may interfere with correct barcode classification.
The output of Dorado trim will always be unaligned records, regardless of whether the
input is aligned/sorted or not.
CLI reference
Positional arguments:
reads Path to a file with reads to trim. Can be in any HTS format.
Required arguments:
-k, --sequencing-kit Sequencing kit name to use for selecting adapters and primers to trim.
Optional arguments:
-h, --help shows help message and exits
-v, --verbose [may be repeated]
-t, --threads Combined number of threads for adapter/primer detection and output generation.
Default uses all available threads.
Input arguments:
-n, --max-reads Maximum number of reads to process.
-l, --read-ids A file with a newline-delimited list of reads to trim.
Output arguments:
--emit-fastq Output in fastq format. Default is BAM.
Main arguments:
--no-trim-primers Skip primer detection and trimming. Only adapters will be detected and trimmed.
--primer-sequences Path to file with custom primer sequences.
Custom primer trimming
Note
Using the --primer-sequences argument will remove the Oxford Nanopore primer sequences from the
trimming search.
Dorado automatically searches for primer sequences used in Oxford Nanopore kits. However, you can specify an alternative set of primer sequences to search for when trimming either in-line with basecalling, or in combination with the --trim option. In both cases this is accomplished using the --primer-sequences command line option. The argument can be either the full path and filename of a FASTA file containing the primer sequences you want to search for, or a string code specifying a supported 3rd-party primer set.
If a FASTA file is specified, then the file must have either the .fa or .fasta extension and must conform to the specification.
The --help option will list the supported 3rd-party primer sets. Currently the only 3rd-party primer set supported is the set of primers used for 10X Genomics sequencing. Support for detecting and trimming these primers can be enabled by using:
--primer-sequences 10X_Genomics
In this case, in addition to detecting and trimming the primers, Dorado will extract the section of the read corresponding to the cell-barcodes and UMI tags and place it in the RX:Z BAM tag.
Effect on demultiplexing
If adapter/primer trimming is done while basecalling in combination with demultiplexing, then Dorado will ensure that the trimming of adapters and primers does not interfere with the demultiplexing process.
For example, trimming will not effect demultiplexing on kit-name in the following command:
However, if you intend to do demultiplexing as a separate step, it is recommended that
trimming is disabled when basecalling with the --no-trim option, to ensure that barcode sequences
remain intact in the calls.