Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics

ABSTRACT Dysbiosis of airway microbiomes has been found in various respiratory diseases, but its molecular details in terms of taxonomic profile, metabolic characteristics, defensive function, and inhabit adaption are far from clear. Shotgun metagenome sequencing provides detailed information for mi...

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Autores principales:	Xing Shi, Changjun Shao, Chunxiong Luo, Yanan Chu, Jian Wang, Qingren Meng, Jun Yu, Zhancheng Gao, Yu Kang
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	emulsion metagenomics microfluidic chip respiratory microbiome Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d4754d193b6545b7b429c5c7916a6e40
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spelling	oai:doaj.org-article:d4754d193b6545b7b429c5c7916a6e402021-12-02T19:46:18ZMicrofluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics10.1128/mSystems.00198-192379-5077https://doaj.org/article/d4754d193b6545b7b429c5c7916a6e402019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00198-19https://doaj.org/toc/2379-5077ABSTRACT Dysbiosis of airway microbiomes has been found in various respiratory diseases, but its molecular details in terms of taxonomic profile, metabolic characteristics, defensive function, and inhabit adaption are far from clear. Shotgun metagenome sequencing provides detailed information for microbes, whereas its application is rather limited in airways due to host DNA contaminants that overwhelm a minute amount of microbial content. Here, we describe a microfluidics-based enrichment device and an emulsion-based whole-genome amplification procedure (MEEA) for the preparation of DNA from sputa for shotgun sequencing in a metagenomics study. The two protocols coupled in MEEA are first separately assayed with mock samples and are both promising in efficiency and bias. The efficiency and consistency of MEEA are further evaluated in six clinical sputum samples against direct sequencing without enrichment, and MEEA enables 2 to 14 times enrichment for microbial reads, which take 14.68% to 33.52% of total reads. The dominant pathogens detected in MEEA are in excellent agreement with those from clinical etiological tests. Meanwhile, MEEA presents much more microbiome complexity and genome information at a strain level than direct sequencing, exhibiting high sensitivity for identifying prophages and DNA viruses. MEEA provides better microbiome profiling than direct sequencing without a preference for specific microorganisms. The more detailed functional and taxonomic characterization of their species constituents, including both bacterium and virus, facilitates metagenomics studies on the pathogenesis of respiratory microbiomes. IMPORTANCE The airway microbial community, which takes important pathogenic roles for respiratory diseases, is far from clear in terms of taxonomy and gene functions. One of the critical reasons is the heavy contamination of host cell/DNA in airway samples, which hinders the subsequent sequencing of the whole genomic contents of the microbial community—the metagenome. Here, we describe a protocol for airway sample preparation which couples a microbe enrichment microfluidic device and a DNA amplification method performed in numerous droplets. When evaluated with mock and clinical sputum samples, the microfluidics-based enrichment device and emulsion-based whole-genome amplification (MEEA) procedure efficiently removes host cells, amplifies the microbial genome, and shows no obvious bias among microbes. The efficiency of MEEA makes it a promising method in research of respiratory microbial communities and their roles in diseases.Xing ShiChangjun ShaoChunxiong LuoYanan ChuJian WangQingren MengJun YuZhancheng GaoYu KangAmerican Society for Microbiologyarticleemulsionmetagenomicsmicrofluidic chiprespiratory microbiomeMicrobiologyQR1-502ENmSystems, Vol 4, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	emulsion metagenomics microfluidic chip respiratory microbiome Microbiology QR1-502
spellingShingle	emulsion metagenomics microfluidic chip respiratory microbiome Microbiology QR1-502 Xing Shi Changjun Shao Chunxiong Luo Yanan Chu Jian Wang Qingren Meng Jun Yu Zhancheng Gao Yu Kang Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics
description	ABSTRACT Dysbiosis of airway microbiomes has been found in various respiratory diseases, but its molecular details in terms of taxonomic profile, metabolic characteristics, defensive function, and inhabit adaption are far from clear. Shotgun metagenome sequencing provides detailed information for microbes, whereas its application is rather limited in airways due to host DNA contaminants that overwhelm a minute amount of microbial content. Here, we describe a microfluidics-based enrichment device and an emulsion-based whole-genome amplification procedure (MEEA) for the preparation of DNA from sputa for shotgun sequencing in a metagenomics study. The two protocols coupled in MEEA are first separately assayed with mock samples and are both promising in efficiency and bias. The efficiency and consistency of MEEA are further evaluated in six clinical sputum samples against direct sequencing without enrichment, and MEEA enables 2 to 14 times enrichment for microbial reads, which take 14.68% to 33.52% of total reads. The dominant pathogens detected in MEEA are in excellent agreement with those from clinical etiological tests. Meanwhile, MEEA presents much more microbiome complexity and genome information at a strain level than direct sequencing, exhibiting high sensitivity for identifying prophages and DNA viruses. MEEA provides better microbiome profiling than direct sequencing without a preference for specific microorganisms. The more detailed functional and taxonomic characterization of their species constituents, including both bacterium and virus, facilitates metagenomics studies on the pathogenesis of respiratory microbiomes. IMPORTANCE The airway microbial community, which takes important pathogenic roles for respiratory diseases, is far from clear in terms of taxonomy and gene functions. One of the critical reasons is the heavy contamination of host cell/DNA in airway samples, which hinders the subsequent sequencing of the whole genomic contents of the microbial community—the metagenome. Here, we describe a protocol for airway sample preparation which couples a microbe enrichment microfluidic device and a DNA amplification method performed in numerous droplets. When evaluated with mock and clinical sputum samples, the microfluidics-based enrichment device and emulsion-based whole-genome amplification (MEEA) procedure efficiently removes host cells, amplifies the microbial genome, and shows no obvious bias among microbes. The efficiency of MEEA makes it a promising method in research of respiratory microbial communities and their roles in diseases.
format	article
author	Xing Shi Changjun Shao Chunxiong Luo Yanan Chu Jian Wang Qingren Meng Jun Yu Zhancheng Gao Yu Kang
author_facet	Xing Shi Changjun Shao Chunxiong Luo Yanan Chu Jian Wang Qingren Meng Jun Yu Zhancheng Gao Yu Kang
author_sort	Xing Shi
title	Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics
title_short	Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics
title_full	Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics
title_fullStr	Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics
title_full_unstemmed	Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics
title_sort	microfluidics-based enrichment and whole-genome amplification enable strain-level resolution for airway metagenomics
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/d4754d193b6545b7b429c5c7916a6e40
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Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway Metagenomics

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