Poly(A) Estimation

Dorado has initial support for estimating poly(A) tail lengths for cDNA (PCS and PCB kits) and RNA, and can be configured for use with custom primer sequences, interrupted tails, and plasmids.

Poly(A) and Poly(T)

Oxford Nanopore cDNA reads are sequenced in two different orientations and Dorado poly(A) tail length estimation handles both (A and T homopolymers).

This feature can be enabled by setting --estimate-poly-a argument which is disabled by default.

The estimated tail length is stored in the pt:i tag of the output record. Reads for which the tail length could not be estimated will have a value of -1 for the pt:i tag if the primer anchor for the tail was not found, or a value of 0 if the primer anchor was found, but the length could not be estimated.

Dorado does not edit the original basecalled sequence using the results of the poly(A/T) estimate.

Custom poly(A) tail configuration

The default settings for this feature are optimized for non-interrupted poly(A/T) sequences that occur at read ends but these setting can be configured using a configuration file which is passed into Dorado using the --poly-a-config argument.

This configuration file can configure parameters for:

Custom primer sequence for cDNA tail estimation
Clustering of interrupted poly(A/T) tails
Estimation of poly(A/T) length in plasmids

Poly(A/T) reference diagram

cDNA

 5' --- ADAPTER --- FRONT_PRIMER
... --- cDNA
... --- poly(A) --- RC(REAR_PRIMER) --- 3'

OR

 5' --- ADAPTER --- REAR_PRIMER  --- poly(T)
... --- RC(cDNA)
... --- RC(FRONT_PRIMER) --- 3'

dRNA

3' --- ADAPTER --- poly(A) --- RNA --- 5'

Plasmid

 5' --- ADAPTER
... --- DNA
... --- FRONT_FLANK --- poly(A) --- REAR_FLANK
... --- DNA --- 3'

OR

 5' --- ADAPTER
... --- RC(DNA)
... --- RC(REAR_FLANK) --- poly(T) --- RC(FRONT_FLANK)
... --- RC(DNA) --- 3'

Configuration format

The poly(A) configuration file uses the toml format.

The content of the file depends on the application i.e. cDNA or plasmids.

cDNAPlasmid

polya_config.cdna.toml

[anchors]
front_primer = "ATCG"
rear_primer = "CGTA"
primer_window = 150

[threshold]
flank_threshold = 0.6

[tail]
tail_interrupt_length = 10

polya_config.plasmid.toml

[anchors]
plasmid_front_flank = "CGATCG"
plasmid_rear_flank = "TGACTGC"
primer_window = 150

[threshold]
flank_threshold = 0.6

[tail]
tail_interrupt_length = 10

Overrides

Configuration options can be overridden for individual barcodes. We generate a default configuration as normal, and then add overrides of specific values for each barcode by adding an [[overrides]] section labelled by the barcode name.

polya_config.toml

[anchors]
front_primer = "ATCG"
rear_primer = "CGTA"
[threshold]
flank_threshold = 0.6
[tail]
tail_interrupt_length = 5

[[overrides]]
barcode_id = "Custom-Kit_barcode01"
[overrides.threshold]
flank_threshold = 0.5       # overrides 0.6

[[overrides]]
barcode_id = "Custom-Kit_barcode02"
[overrides.anchors]
front_primer = "AACC"       # overrides ATCG
rear_primer = "GGTT"        # overrides CGTA
[overrides.tail]
tail_interrupt_length = 10  # overrides 5

This creates three configurations:

a default configuration with custom front and rear primers and an interrupt length of 5
a configuration to use for barcode01 from kit Custom-Kit almost identical to the main custom settings (i.e. with the custom front and rear primers and the interrupt length), with an additional change to the flank_threshold.
a configuration to use for barcode02 from kit Custom-Kit with different primers and an interrupt length of 10, but with no change to the flank threshold.

Configuration options

Config Group	Option	Description
anchors	front_primer	Front primer sequence for cDNA^[1]
anchors	rear_primer	Rear primer sequence for cDNA^[1]
anchors	plasmid_front_flank	Front flanking sequence of poly(A) in plasmid^[2]
anchors	plasmid_rear_flank	Rear flanking sequence of poly(A) in plasmid^[2]
anchors	primer_window	Window of bases at the front and rear of the rear within which to look for primer sequences
threshold	flank_threshold	Threshold to use for detection of the flank/primer sequences. Equates to `(1 - edit distance / flank_sequence)`
tail	tail_interrupt_length	Combine tails that are within this distance of each other (default is 0, i.e. don't combine any)

For cDNA only - Values ignored if either plasmid_front_flank or plasmid_rear_flank are set.
For plasmids only