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PolyTailConfig.md

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Custom Poly Tail Configuration

Dorado supports estimation of Poly(A/T) tails for DNA (PCS and PCB) and RNA samples. The default settings are optimized for non-interrupted Poly(A/T) sequences that occur at read ends.

Dorado also supports additional features that can be customized through a configuration file (described below):

  • 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 configuration file needs to be in the toml format.

[anchors]
front_primer = "ATCG"
rear_primer = "CGTA"
plasmid_front_flank = "CGATCG"
plasmid_rear_flank = "TGACTGC"

[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.

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

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

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

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 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

Option Description
front_primer Front primer sequence for cDNA
rear_primer Rear primer sequence for cDNA
plasmid_front_flank Front flanking sequence of poly(A) in plasmid
plasmid_rear_flank Rear flanking sequence of poly(A) in plasmid
flank_threshold Threshold to use for detection of the flank/primer sequences. Equates to (1 - edit distance / flank_sequence) (default: 0.6)
primer_window Window of bases at the front and rear of the rear within which to look for primer sequences (default: 150)
min_primer_separation Minimum difference in edit distance between the forward and reverse alignments of the primers required in order to proceed with estimation (default: 10)
tail_interrupt_length Combine tails that are within this distance of each other (default: 0, i.e. don't combine any)