Getting Started with VigyanLLM
Design lab-ready primer and probe pairs with our 24-step biophysical validation pipeline. From sequence input to an audit-ready PDF report in minutes.
How the Pipeline Works
VigyanLLM's pipeline is a fully automated 24-step primer and probe design engine. It takes a DNA sequence or gene name as input and produces lab-ready primer pairs with an audit-grade PDF report. The entire pipeline runs server-side — the browser submits the job and polls for results.
Architecture Overview
When you submit a target sequence, the job is dispatched to a Flask backend running Primer3, NCBI BLAST, and the SantaLucia nearest-neighbour thermodynamics engine. Each of the 24-steps is executed sequentially. If any step fails (e.g., BLAST is unreachable), the pipeline marks that job as failed and returns a partial result. Results are stored server-side and fetched by the frontend via polling.
Pipeline at a Glance
Phase A
Target Selection & Filtering
Phase B
Primer Design & Thermodynamics
Phase C
Specificity & Alignment
Phase D
Secondary Structure & Validation
Phase E
Ranking & Report
Step-by-Step Pipeline Flow
| Step | Phase | Description |
|---|---|---|
| 1 | A | Fetch reference transcript & genomic sequence from NCBI |
| 2 | A | Filter low-complexity & repeat-masked regions |
| 3 | A | Identify conserved & unique regions for primer placement |
| 4 | A | Exclude known SNP-dense regions from target window |
| 5 | A | Select optimal amplification window based on GC profile |
| 6 | B | Run Primer3 with configured Tm, length, and GC% parameters |
| 7 | B | Apply 3′ GC clamp & end-stability filters |
| 8 | B | Calculate Tm via SantaLucia 1998 nearest-neighbour model |
| 9 | B | Enforce ΔTm ≤ 1.5°C between forward & reverse |
| 10 | C | BLAST each candidate against reference genome (GRCh38/hg38) |
| 11 | C | Filter off-targets with ≤ 2 mismatches in last 5 bp at 3′ end |
| 12 | C | Cross-reference amplicon against known pseudogenes |
| 13 | D | Predict hairpin secondary structure (ΔG > −2.0 kcal/mol) |
| 14 | D | Predict self-dimer formation (ΔG > −8.0 kcal/mol) |
| 15 | D | Predict cross-dimer between Fwd & Rev (ΔG > −8.0 kcal/mol) |
| 16 | D | Mask primers overlapping with Dfam repeat elements |
| 17 | D | Cross-reference positions against dbSNP common variants |
| 18 | D | Evaluate multiplex compatibility (cross-pair ΔG > −5.0) |
| 19 | D | Design TaqMan probe (if enabled): Tm 68–70°C, no G at 5′ |
| 20 | E | Compute composite score (0–100) per primer pair |
| 21 | E | Rank pairs & filter to top 10 best candidates |
| 22 | E | Generate PDF audit report with full traceability |
How Scoring Works
Each of the 24-steps contributes a weighted sub-score to the final 0–100 rating. Thermodynamic penalties (hairpin, self-dimer, cross-dimer) carry the heaviest weight because they directly affect PCR efficiency. BLAST off-target count and SNP overlap also reduce the score. Pairs scoring 80+ are marked green (lab-ready), 60–79 are yellow (requires verification), 40–59 are orange (use with caution), and <40 are red (discard).
User Workflow
1Enter Your Sequence
Paste a DNA template sequence or a gene name (e.g., TP53, BRCA1). The pipeline auto-fetches the transcript from NCBI. Supported length: 50–10,000 bp.
2Configure Parameters
Set primer Tm (default 62°C), amplicon size range, and design mode (Standard PCR, Bisulfite MSP, or Multiplex). Enable TaqMan probe design or NGS adapter tailing as needed.
Optional3Run Pipeline
Click Run Pipeline. The 24-step engine executes server-side in sequence. The frontend polls for progress and updates the UI as each phase completes. Typical runtime: 30–60 seconds depending on target length and server load.
~30–60 seconds4Review Results
Each primer pair is scored 0–100. Green (80+) pairs are lab-ready. Yellow/orange pairs need verification. Red pairs should be discarded. Expand any row to see full metrics: Tm, GC%, hairpin ΔG, self-dimer ΔG, cross-dimer ΔG, and BLAST off-target hits.
5Export & Report
Download results as CSV, JSON, or a comprehensive PDF audit report. Order-ready reports for IDT and Twist Bioscience are also available for direct synthesis ordering.
PDF includes full audit trailTry the Pipeline Now
No account required for the demo. Enter a sequence and see the 24-step engine in action.