VIRUSSPRIMERPRO

Conserved Primer Pair Design at Viral Species and Subspecies Level

Step 1

Upload Input Data

Upload an aligned FASTA as the core input. Add a GenBank file if you want annotation context in the result viewer.

FASTA / FAS / FA GenBank Optional Tree Optional

Required Primary alignment source

Aligned Genome MSA

Choose FASTA file

or drag and drop here

Accepted formats: FASTA, FAS, FA

Optional Used by the annotation bar in the result viewer

GenBank Annotation

Choose GenBank file

or drag and drop here

Must match the first sequence in the uploaded alignment.

Optional Used for notifications or manual follow-up

Notification Email

Step 2

Choose A Design Mode

Pick the workflow that matches your sequence diversity. The configuration panel below updates immediately for the selected engine.

Conserved Region Traditional mode for low-variation genes

Primer Site Advanced mode for high-variation genes

Phylo Guide Tree-guided design for a user-defined clade

Step 3

Tune Design Parameters

Use defaults for a quick run, or refine thresholds, search strategy, and downstream behavior before submission.

Configuration Parameters

Settings for conserved region detection and primer candidate generation

Conserved Region Detection

These parameters determine how conserved regions are defined before primer candidates are generated.

Min Continuous Length (bp) Default: 10

Paired Site Max Diff Default: 0.05

Max Tolerance Default: 1

Primer Candidate Generation

These parameters control candidate primer generation before the result page. The result page only filters and pairs generated primers.

Candidate Primer Min Length (bp) 15-25

Candidate Primer Max Length (bp) 15-25

Na+ Concentration (M) Default: 0.05

Configuration Parameters

Advanced settings for primer site mode

Basic Parameters

Primer Min Length (bp) 15-25

Primer Max Length (bp) 15-25

Window Scoring Parameters

Max Gap Rate 0-1, Default: 0.1

Min Valid Rate 0-1, Default: 0.9

Body P-Max Threshold 0-1, Default: 0.8

3' End P-Max Threshold 0-1, Default: 0.9

3' End Window Size (bp) Default: 5

Max Entropy (optional) Leave empty to disable

Scoring Weights

3' End Weight Default: 0.5

Body Weight Default: 0.3

Gap Penalty Weight Default: 0.2

Degenerate Primer Strategy

Strategy

Max Degeneracy Default: 16

No Degenerate 3' Length (bp) Default: 5

Target Coverage 0-1, Default: 0.95

Degenerate Main Search

Controls whether IUPAC degenerate candidates enter the v3 main search, not just the legacy post-processing reports.

Search Mode Default: Off

Degenerate Frequency Threshold Default: 0.95

Max Degenerate Candidates / Window Default: 1

Degenerate Rank Penalty Default: 0.06

Fallback Min Best Pair Coverage Optional, blank = use target coverage

v3 Coverage & Pairing Tuning

Strict Bind Threshold Default: 3.0

Relaxed Bind Threshold Default: 4.0

Strict Tail Window (bp) Default: 3

Pareto Pool Size Default: 300

Pareto Max Per Start Default: 5

Top Primers For Pairing Default: 180

Max Evaluated Pairs Default: 5000

Amplicon Min (bp) Default: 120

Amplicon Max (bp) Default: 1200

Rare Cluster Floor Default: 0.60

Min Cluster Floor Default: 0.70

Reference Sequence Strategy

Reference Strategy

Tie Breaker (for Consensus)

1. Upload 2. Validate 3. Annotate 4. Review & Submit

Upload an aligned FASTA and a matching Newick tree to start validation.

MSA IDs

Tree Leaves

Matched

Missing In Tree

Validation has not started.

Matched IDs

Missing In Tree

Extra In Tree

Repeated FASTA IDs

Repeated Tree IDs

Tree Annotation

Click to single-select, use Cmd/Ctrl/Shift-click for multi-select, or drag on the tree to box-select leaf rows before assigning them into genotype, background, or excluded groups.

Search Sequence IDs Tree Length Scale

1.6x

Root Controls

Enable reroot

No node selected.

Current Selection

none

Click to single-select, Cmd/Ctrl/Shift-click to add or remove items, or drag on the tree to box-select leaf rows.

Reference Genotype Import

Upload a CSV/TSV or paste rows like seq_id,genotype or tree_name,genotype to batch assign known reference genomes.

Reference Mapping File

Paste Reference Rows

No reference genotype import applied yet.

Genotype Name

Tip: plain click replaces selection, Cmd/Ctrl/Shift-click toggles, drag on the tree box-selects leaves.

Level Annotation Shortcuts Uses the primary selected node's genotype, or the genotype name input above.

Group Summary

Unassigned: 0

Sequence List

0 shown

Step 4

Run The Design Job

Submit the current dataset and configuration. The progress panel below will track the computational stages and expose the final result links when the job finishes.

Primer Pools

VIRUSSPRIMERPRO

Upload Input Data

Choose A Design Mode

Tune Design Parameters

Configuration Parameters

Conserved Region Detection

Primer Candidate Generation

Configuration Parameters

Basic Parameters

Degenerate Main Search

Phylo Guide Configuration

Matched IDs

Missing In Tree

Extra In Tree

Repeated FASTA IDs

Repeated Tree IDs

Name Match Review

Tree Annotation

Current Selection

Reference Genotype Import

Group Summary

Sequence List

Review & Submit

Run The Design Job