Dual Barcoded Shotgun Expression Library for Microbial Trait Characterization
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Solution Overview
Problem
Current technologies face challenges in characterizing the functions of uncultivated microbes and amplified genomes from single-cell genomics due to limitations in high-throughput, scalability, and cost-effectiveness, particularly in inferring gene functions and conducting gain-of-function assays across diverse conditions.
Innovation Solution
The development of a dual barcoded shotgun expression library (Dub-seq) technology, which involves constructing expression libraries with nucleic acid fragments flanked by barcodes, transforming them into host organisms, and subjecting them to stress conditions to identify resistant fragments, allowing for quantitative analysis and multiplex tracking of microbial fitness across hundreds of conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current shotgun expression library methods are used to characterize uncultivated microbes, then gene function information can be obtained, but the process is low throughput, expensive, and labor-intensive
Solution Approach 1:
The patent combines multiple characterization steps into a single dual-barcode library construction and sequencing workflow. By integrating both barcodes (first and second) into one library preparation process and using paired-end sequencing to capture both barcodes simultaneously, the method merges library construction, barcode assignment, and sample identification into a unified high-throughput pipeline that eliminates repetitive manual operations.
Solution Approach 2:
The patent uses barcode sequences as digital copies to represent and track individual nucleic acid fragments throughout the experimental process. The dual-barcode system creates unique digital identifiers for each fragment, allowing high-throughput tracking and quantification without requiring physical manipulation of individual samples, thereby dramatically increasing throughput while maintaining characterization precision.
2Loss of information
If shotgun expression libraries are constructed to perform gain-of-function assays, then functional information can be discovered, but the process is expensive and labor-intensive
Solution Approach 1:
The dual-barcode library construction method serves multiple functions simultaneously: it enables library construction, assigns unique identifiers to fragments, allows multiplexed tracking across multiple conditions, and provides quantitative measurement capability. This universal approach replaces multiple separate procedures with a single integrated workflow, reducing both cost and labor while preserving functional information discovery.
Solution Approach 2:
The patent changes the parameter of identification from single-barcode to dual-barcode system, and from qualitative to quantitative measurement. By using two barcodes per fragment and sequencing both, the method enables precise quantification of fragment abundance across multiple conditions, providing comprehensive functional information while streamlining the manufacturing process through standardized library preparation.
3Measurement precision
If expression libraries are transformed into host organisms for functional screening, then gene functions can be characterized, but scalability to hundreds of conditions is limited
Solution Approach 1:
The dual-barcode system creates digital copies that can be tracked across unlimited conditions without additional physical manipulation. Each nucleic acid fragment's fate across hundreds of different screening conditions is recorded as changes in barcode sequence abundance, enabling scalable analysis of functional characteristics across diverse conditions while maintaining measurement precision through sequencing-based quantification.
Solution Approach 2:
The patent replaces mechanical tracking methods (physical sorting, individual sample handling) with sequencing-based detection of barcode sequences. This substitution allows simultaneous monitoring of fragment abundance across hundreds of conditions through a single sequencing run, dramatically increasing scalability while preserving functional characterization accuracy through quantitative sequence analysis.
Data Source
AI summary
Disclosed herein are barcoded expression libraries comprising a plurality of expression vectors, wherein each expression vector comprises a nucleic acid fragment flanked by a first barcode and a second barcode. Further disclosed herein are methods of making the barcoded expression libraries and methods of conducting functional analysis using the barcoded expression libraries.