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

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CleanBar: a versatile demultiplexing tool for split-and-pool barcoding in single-cell omics.

Vicente Arnau¹, Alicia Ortiz-Maiques¹, Juan Valero-Tebar¹

¹Institute for Integrative Systems Biology (I2SysBio), University of Valencia and Spanish National Research Council (CSIC), 46980, Paterna, Valencia, Spain.

ISME Communications

|August 27, 2025

Summary

A new bioinformatic tool, CleanBar, enables accurate demultiplexing of single cells barcoded using split-and-pool methods. This advances microbial single-cell genomics and the study of phage-bacteria interactions.

Keywords:

Atrandi PacBio demultiplexing microbial single-cell genomics phage-bacteria interactions split-and-pool barcoding

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Area of Science:

Microbial Ecology
Genomics
Bioinformatics

Background:

Split-and-pool barcoding is crucial for single-cell omics, but lacks a universal bioinformatic tool for demultiplexing.
Existing methods struggle with variations in barcode position, linker length, and ligation errors.

Purpose of the Study:

To develop a flexible and accurate bioinformatic tool for demultiplexing single cells barcoded with sequential ligations.
To address limitations in current tools for analyzing microbial single-cell genomics data.

Main Methods:

Development of CleanBar, a novel bioinformatic tool for demultiplexing sequentially ligated barcodes.
Application of CleanBar with the Atrandi platform and PacBio sequencing for microbial single-cell genomics.
Testing CleanBar's ability to handle barcode variations and ligation errors.

Main Results:

CleanBar accurately demultiplexes reads, accommodating variations in barcode positions and linker lengths.
The tool successfully prevents misclassification of natural barcode-like sequences and handles ligation errors.
Single-cell genomics of *Klebsiella* strains infected with phages revealed infection heterogeneity and enabled phage copy number estimation.

Conclusions:

CleanBar is an efficient, adaptable, and precise tool for microbial single-cell genomics.
Combined with the Atrandi platform and PacBio sequencing, CleanBar enhances high-throughput analysis of microbial ecology and evolution.
The tool facilitates deeper understanding of phage-bacteria interactions at the single-cell level.