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

Nat Commun. 2019 Dec 16;10(1):5729. doi: 10.1038/s41467-019-13719-9.


While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation. We determine that 1-3% of mutants in Streptococcus pneumoniae have a different fitness when grown in isolation and show how dTn-Seq can help identify leads for gene function, including those involved in hyper-competence, processing of alpha-1-acid glycoprotein, sensitivity against the human leukocyte elastase and microcolony formation. Additionally, we show dTn-Seq compatibility with microscopy, FACS and investigations of bacterial cell-to-cell and bacteria-host cell interactions. dTn-Seq reduces costs and retains the advantages of Tn-Seq, while expanding the method's original applicability.

Publication types

  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • DNA Transposable Elements / genetics*
  • DNA, Bacterial / genetics
  • Gene Library
  • Genes, Bacterial / genetics
  • High-Throughput Nucleotide Sequencing / instrumentation
  • High-Throughput Nucleotide Sequencing / methods*
  • Microfluidic Analytical Techniques / instrumentation
  • Microfluidic Analytical Techniques / methods*
  • Mutation
  • Sequence Analysis, DNA / methods*
  • Single-Cell Analysis / instrumentation
  • Single-Cell Analysis / methods*
  • Streptococcus pneumoniae / genetics


  • DNA Transposable Elements
  • DNA, Bacterial