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Curated Papers > Recent high-impact research on "single-cell sequencing" (IF ≥ 30, last 5 years)

Massively parallel single-cell sequencing of diverse microbial populations. ​

Literature Information ​

DOI10.1038/s41592-023-02157-7
PMID38233503
JournalNature methods
Impact Factor32.1
JCR QuartileQ1
Publication Year2024
Times Cited13
Keywordssingle-cell sequencing, microbial populations, antibiotic resistance genes
Literature TypeJournal Article
ISSN1548-7091
Pages228-235
Issue21(2)
AuthorsFreeman Lan, Jason Saba, Tyler D Ross, Zhichao Zhou, Katie Krauska, Karthik Anantharaman, Robert Landick, Ophelia S Venturelli

TL;DR ​

This study introduces DoTA-seq, a novel droplet microfluidics-based method for high-throughput single-cell sequencing, enabling the analysis of genetic heterogeneity in microbial populations. The method successfully tracks antibiotic resistance genes and plasmids in gut microbiomes, offering a powerful tool for microbial genetic research and enhancing our understanding of microbial biology.

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single-cell sequencing · microbial populations · antibiotic resistance genes

Abstract ​

Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.

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Primary Questions Addressed ​

  1. What are the specific advantages of using droplet microfluidics in DoTA-seq compared to traditional sequencing methods?
  2. How does DoTA-seq facilitate the study of antibiotic resistance genes in different microbial environments?
  3. In what ways can the findings from DoTA-seq be applied to improve our understanding of microbial interactions in the gut?
  4. What challenges might researchers face when implementing DoTA-seq in diverse microbial populations, and how can these be overcome?
  5. How does the genetic heterogeneity observed in microbial populations impact their ecological roles and functions?

Key Findings ​

Research Background and Objectives ​

Microbial populations exhibit significant genetic heterogeneity at the single-cell level, which is crucial for understanding microbial biology and ecology. However, existing methods to study this heterogeneity are often limited in accessibility and applicability. This study aims to develop a robust, high-throughput method for single-cell sequencing of target genetic loci in various microbial species, addressing the need for a more efficient approach to analyze genetic diversity within microbial communities.

Main Methods/Materials/Experimental Design ​

The study introduces a novel technique called droplet targeted amplicon sequencing (DoTA-seq), which utilizes droplet microfluidics to enable high-throughput single-cell sequencing. The following steps outline the key components of the experimental design:

Mermaid diagram
  1. Sample Collection: Samples are obtained from human and mouse gut microbial communities.
  2. Cell Dispersion: The microbial cells are dispersed to ensure single-cell isolation.
  3. Droplet Generation: A microfluidic device generates droplets containing individual cells.
  4. Target Locus Amplification: Specific genetic loci (e.g., antibiotic-resistance genes) are amplified within each droplet.
  5. Sequencing Library Preparation: The amplified products are prepared for sequencing.
  6. Sequencing: High-throughput sequencing is performed to analyze the genetic material.
  7. Data Analysis: The sequencing data is analyzed to assess the prevalence and taxonomic associations of the target genes.

Key Results and Findings ​

  • DoTA-seq successfully tracked over 10 antibiotic-resistance genes and plasmids across diverse microbial populations from human and mouse gut samples.
  • The method demonstrated high sensitivity and specificity, allowing for the identification of genetic variations at the single-cell level.
  • The results highlighted significant genetic diversity within microbial communities, with varying prevalence of resistance genes linked to specific taxa.

Main Conclusions/Significance/Innovation ​

The development of DoTA-seq represents a significant advancement in the field of microbial genetics. This method provides a simple and accessible platform for high-throughput single-cell sequencing, enabling researchers to:

  • Explore genetic heterogeneity in microbial populations more efficiently.
  • Investigate the relationships between genetic traits and microbial community dynamics.
  • Assess the implications of antibiotic resistance within complex microbial ecosystems.

Research Limitations and Future Directions ​

While DoTA-seq offers numerous advantages, some limitations and future research directions include:

LimitationsFuture Directions
Potential bias in droplet formation affecting cell representationExplore optimization of droplet generation techniques
Limited to specific genetic loci targeted for amplificationExpand the method to include whole-genome sequencing for broader applications
Data analysis complexity due to high-dimensional datasetsDevelop more advanced computational tools for data interpretation

Future research should focus on refining the DoTA-seq technique, enhancing its application scope, and integrating it with other genomic technologies to provide a more comprehensive understanding of microbial diversity and function.

References ​

  1. Proteomic Investigation of Tolerant Escherichia coli Populations from Cyclic Antibiotic Treatment. - Jordy Evan Sulaiman;Henry Lam - Journal of proteome research (2020)
  2. A Subset of Polysaccharide Capsules in the Human Symbiont Bacteroides thetaiotaomicron Promote Increased Competitive Fitness in the Mouse Gut. - Nathan T Porter;Pablo Canales;Daniel A Peterson;Eric C Martens - Cell host & microbe (2017)
  3. Sequence changes in the pilus subunit lead to tropism variation of Neisseria gonorrhoeae to human tissue. - A B Jonsson;D Ilver;P Falk;J Pepose;S Normark - Molecular microbiology (1994)
  4. Epigenetic Switch Driven by DNA Inversions Dictates Phase Variation in Streptococcus pneumoniae. - Jing Li;Jing-Wen Li;Zhixing Feng;Juanjuan Wang;Haoran An;Yanni Liu;Yang Wang;Kailing Wang;Xuegong Zhang;Zhun Miao;Wenbo Liang;Robert Sebra;Guilin Wang;Wen-Ching Wang;Jing-Ren Zhang - PLoS pathogens (2016)
  5. Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. - Yann Marcy;Cleber Ouverney;Elisabeth M Bik;Tina Lösekann;Natalia Ivanova;Hector Garcia Martin;Ernest Szeto;Darren Platt;Philip Hugenholtz;David A Relman;Stephen R Quake - Proceedings of the National Academy of Sciences of the United States of America (2007)
  6. Insights into the phylogeny and coding potential of microbial dark matter. - Christian Rinke;Patrick Schwientek;Alexander Sczyrba;Natalia N Ivanova;Iain J Anderson;Jan-Fang Cheng;Aaron Darling;Stephanie Malfatti;Brandon K Swan;Esther A Gies;Jeremy A Dodsworth;Brian P Hedlund;George Tsiamis;Stefan M Sievert;Wen-Tso Liu;Jonathan A Eisen;Steven J Hallam;Nikos C Kyrpides;Ramunas Stepanauskas;Edward M Rubin;Philip Hugenholtz;Tanja Woyke - Nature (2013)
  7. Single-cell analysis of multiple invertible promoters reveals differential inversion rates as a strong determinant of bacterial population heterogeneity. - Freeman Lan;Jason Saba;Yili Qian;Tyler Ross;Robert Landick;Ophelia S Venturelli - Science advances (2023)
  8. Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing. - Sydney B Blattman;Wenyan Jiang;Panos Oikonomou;Saeed Tavazoie - Nature microbiology (2020)
  9. Microbial single-cell RNA sequencing by split-pool barcoding. - Anna Kuchina;Leandra M Brettner;Luana Paleologu;Charles M Roco;Alexander B Rosenberg;Alberto Carignano;Ryan Kibler;Matthew Hirano;R William DePaolo;Georg Seelig - Science (New York, N.Y.) (2021)
  10. Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding. - Freeman Lan;Benjamin Demaree;Noorsher Ahmed;Adam R Abate - Nature biotechnology (2017)

Literatures Citing This Work ​

  1. Single-cell analysis of multiple invertible promoters reveals differential inversion rates as a strong determinant of bacterial population heterogeneity. - Freeman Lan;Jason Saba;Yili Qian;Tyler Ross;Robert Landick;Ophelia S Venturelli - Science advances (2023)
  2. Integrating evolutionary genomics of forest trees to inform future tree breeding amid rapid climate change. - Jiajun Feng;Xuming Dan;Yangkai Cui;Yi Gong;Minyue Peng;Yupeng Sang;Pär K Ingvarsson;Jing Wang - Plant communications (2024)
  3. AI in microbiome-related healthcare. - Niklas Probul;Zihua Huang;Christina Caroline Saak;Jan Baumbach;Markus List - Microbial biotechnology (2024)
  4. Microbial functional diversity and redundancy: moving forward. - Pierre Ramond;Pierre E Galand;Ramiro Logares - FEMS microbiology reviews (2025)
  5. Bacteroides expand the functional versatility of a conserved transcription factor and transcribed DNA to program capsule diversity. - Jason Saba;Katia Flores;Bailey Marshall;Michael D Engstrom;Yikai Peng;Atharv S Garje;Laurie E Comstock;Robert Landick - Nature communications (2024)
  6. A data-driven modeling framework for mapping genotypes to synthetic microbial community functions. - Yili Qian;Sarvesh D Menon;Nick Quinn-Bohmann;Sean M Gibbons;Ophelia S Venturelli - bioRxiv : the preprint server for biology (2025)
  7. Self-driving laboratories, advanced immunotherapies and five more technologies to watch in 2025. - Michael Eisenstein - Nature (2025)
  8. High throughput single cell metagenomic sequencing with semi-permeable capsules: unraveling microbial diversity at the single-cell level in sewage and fecal microbiomes. - Meilee Ling;Judit Szarvas;Vaida Kurmauskaitė;Vaidotas Kiseliovas;Rapolas Žilionis;Baptiste Avot;Patrick Munk;Frank M Aarestrup - Frontiers in microbiology (2024)
  9. Barcodes based on nucleic acid sequences: Applications and challenges (Review). - Ying Hong Wei;Faquan Lin - Molecular medicine reports (2025)
  10. Microbiome Single Cell Atlases Generated with a Commercial Instrument. - Xiangpeng Li;Linfeng Xu;Benjamin Demaree;Cecilia Noecker;Jordan E Bisanz;Daniel W Weisgerber;Cyrus Modavi;Peter J Turnbaugh;Adam R Abate - Advanced science (Weinheim, Baden-Wurttemberg, Germany) (2025)

... (3 more literatures)

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