Hybrid metagenomic assembly platform for high-throughput identification of novel bacillus thuringiensis insecticidal genes

Overview

This platform addresses a critical bottleneck in microbial pesticide discovery: the difficulty of identifying new insecticidal genes from mixed Bacillus thuringiensis (Bt) samples due to the high homology among known insecticidal genes. By combining Nanopore long-read sequencing with second-generation sequencing correction in a hybrid metagenomic assembly workflow, the technology enables high-resolution analysis of highly homologous gene families and significantly improves the efficiency of novel insecticidal gene screening from environmental and strain library samples.

The approach is particularly valuable for agricultural biotechnology companies, biocontrol product developers, and crop protection researchers seeking new modes of action for insect resistance management. Initial validation demonstrated the ability to assemble complete full-length insecticidal gene sequences from complex mixed plasmid samples with exceptional accuracy, including the discovery of three previously unknown vip genes with homology below 60% compared to any known gene.

Technical specifications

Core methodology:

• Hybrid metagenomic assembly combining Nanopore GridION X5 long-read sequencing with second-generation sequencing data for error correction
• Designed to resolve highly homologous insecticidal gene families that confound standard short-read sequencing approaches
• Bioinformatics pipeline optimized for mixed-sample plasmid analysis and full-length gene reconstruction

Validated performance:

• Assembled 95 complete full-length insecticidal genes across 24 gene classes from 25 mixed Bt strain plasmids in a single high-throughput run
• Internal control genes from the standard Bti reference strain achieved 99.99% sequencing accuracy
• Successfully identified three novel vip genes exhibiting less than 60% homology to any previously characterized gene

Development roadmap:

• Algorithm refinement to further improve Nanopore-based gene identification accuracy
• Expansion of the workflow to include macro-plasmid sequencing from both Bt strain libraries and environmental samples
• Establishment of a cost-effective, high-throughput gene screening system for routine use

Technology readiness level

The platform has been experimentally validated using mixed plasmid samples from 25 Bt strains sequenced on the Nanopore GridION X5 platform, demonstrating robust gene assembly and the ability to discover genuinely novel insecticidal genes. The current readiness level reflects a working laboratory-validated workflow with demonstrated proof-of-concept results. Ongoing development efforts focus on optimizing the bioinformatics algorithms and extending the methodology to broader sample types, including environmental isolates. The team is seeking funding support for key personnel to advance the system toward a fully operational high-throughput screening service.