Introduction

Overview

Comprehensive introduction to the MinION pathogen screening pipeline for xenotransplantation.

What is the MinION Pipeline?

The MinION Pathogen Screening Pipeline is a PMDA-compliant metagenomic analysis system designed for comprehensive pathogen detection in donor pigs intended for xenotransplantation. The system uses Oxford Nanopore MinION Mk1D long-read sequencing technology combined with AWS cloud infrastructure for scalable, cost-effective analysis.

This pipeline provides end-to-end automation from basecalling to reporting, ensuring complete coverage of all 91 PMDA-designated pathogens with special emphasis on critical PERV (Porcine Endogenous Retrovirus) detection.

PMDA Compliant

TRUE 100% coverage of all 91 designated pathogens including circular and ssDNA viruses (v2.1)

Cloud-Native

Serverless architecture on AWS with Lambda + EC2 on-demand

Real-time Analysis

Streaming analysis capability with MinION sequencing

Key Features

Q30+ Accuracy

Duplex basecalling with Dorado achieves 99.9% accuracy (Q30+) by sequencing both DNA strands.

Simplex mode: 95% accuracy (Q12-15)
Duplex mode: 99.9% accuracy (Q30+)

Multi-Database Search

Comprehensive pathogen detection using multiple complementary databases:

  • • Kraken2: Rapid taxonomic classification (Standard DB ~50GB)
  • • RVDB v30.0: Manually curated viral sequences (~10GB)
  • • BLAST: PMDA custom database (91 pathogens, ~5GB)
  • • Diamond: Protein-level viral search
  • • PERV-specific: Targeted detection and typing

Automated Workflow

Six automated phases from raw data to PMDA-compliant reports:

1
Basecalling (2-4 hours)
2
Quality Control (10-15 min)
3
Host Removal (30-60 min)
4
Pathogen Detection (1-2 hours)
5
Quantification (15-30 min)
6
Reporting (10-15 min)

Use Cases

Xenotransplantation Research

Primary use case for screening donor pigs before organ transplantation to humans.

  • ✓ Pre-transplant donor screening
  • ✓ Quarterly monitoring of breeding colony
  • ✓ Clinical trial donor qualification
  • ✓ Post-transplant monitoring

Pathogen Surveillance

Broader applications in veterinary and agricultural settings.

  • ✓ Outbreak investigation
  • ✓ Breeding facility monitoring
  • ✓ Biosecurity assessment
  • ✓ Research animal screening

System Requirements

Hardware

Sequencing

  • • Oxford Nanopore MinION Mk1D
  • • R10.4.1 flow cells
  • • Laptop/desktop for MinKNOW

AWS Resources

  • • g4dn.xlarge (GPU basecalling)
  • • r5.4xlarge (pathogen detection)
  • • t3.large/xlarge (general analysis)

Software Dependencies

Basecalling

  • • Dorado v0.5+
  • • CUDA 11.8+
  • • PycoQC
  • • NanoPlot

Alignment

  • • Minimap2
  • • SAMtools
  • • BWA-MEM

Detection

  • • Kraken2/Bracken
  • • BLAST+
  • • Diamond

Performance Metrics

Total Duration

4-8 hrs

Complete analysis pipeline

Accuracy

99.9%

Duplex basecalling (Q30+)

Sensitivity

>95%

For PMDA pathogens

Cost per Run

~$50

AWS compute costs

Next Steps

Quick Start

Get started with deployment and configuration

Follow our step-by-step guide to deploy the pipeline to AWS and run your first analysis.

Architecture Deep Dive

Learn about system design and components

Explore the cloud-native architecture and understand how each component works together.

API Integration

Integrate with your existing systems

Use our REST API to automate workflow execution and retrieve results programmatically.

PMDA Compliance

Understand regulatory requirements

Review the 91 designated pathogens and PERV-specific detection requirements.