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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Published on: May 23, 2018

Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes.

Derek Thibault¹, Paul A Jensen^1,2, Stephen Wood¹

¹Biology Department, Boston College, Chestnut Hill, MA, 02467, USA.

Nature Communications

|December 18, 2019

Summary

Droplet Tn-Seq (dTn-Seq) enables isolated bacterial growth in droplets, revealing single-cell phenotypes masked by pooled cultures. This method identifies novel gene functions and interactions, enhancing bacterial fitness studies.

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Area of Science:

Microbiology
Molecular Biology
Biotechnology

Background:

High-throughput bacterial fitness studies using Transposon sequencing (Tn-Seq) can obscure individual cell behaviors.
Pooled library culturing in Tn-Seq limits the observation of complex single-cell phenotypes.

Purpose of the Study:

To introduce and validate Droplet Tn-Seq (dTn-Seq) for isolated bacterial mutant growth.
To demonstrate dTn-Seq's utility in identifying gene functions and complex phenotypes.
To showcase dTn-Seq's compatibility with various analytical techniques.

Main Methods:

Microfluidic chip design, production, and optimization for droplet encapsulation.
Encapsulation of individual transposon mutants in growth medium-in-oil droplets.
Sample preparation and analysis for dTn-Seq.

Main Results:

1-3% of Streptococcus pneumoniae mutants exhibit altered fitness when grown in isolation.
dTn-Seq identified genes involved in hyper-competence, host-pathogen interactions, and microcolony formation.
Demonstrated compatibility of dTn-Seq with microscopy, FACS, and cell interaction studies.

Conclusions:

dTn-Seq overcomes limitations of traditional Tn-Seq by enabling isolated growth.
The method is cost-effective and expands the applicability of Tn-Seq for bacterial research.
dTn-Seq facilitates discovery of gene functions and complex cellular behaviors.