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...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2019-08-01
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| Series: | mSystems |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSystems.00198-19 |
| _version_ | 1818461181368598528 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T23:42:04Z |
| format | Article |
| id | doaj.art-d4754d193b6545b7b429c5c7916a6e40 |
| institution | Directory Open Access Journal |
| issn | 2379-5077 |
| language | English |
| last_indexed | 2024-12-14T23:42:04Z |
| publishDate | 2019-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSystems |
| spelling | doaj.art-d4754d193b6545b7b429c5c7916a6e402022-12-21T22:43:30ZengAmerican Society for MicrobiologymSystems2379-50772019-08-014410.1128/mSystems.00198-19Microfluidics-Based Enrichment and Whole-Genome Amplification Enable Strain-Level Resolution for Airway MetagenomicsXing Shi0Changjun Shao1Chunxiong Luo2Yanan Chu3Jian Wang4Qingren Meng5Jun Yu6Zhancheng Gao7Yu Kang8CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaCAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaThe State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, School of Physics, Peking University, Beijing, People’s Republic of ChinaCAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaCAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaCAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaCAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaDepartment of Respiratory and Critical Care Medicine, Peking University People’s Hospital, Beijing, People’s Republic of ChinaCAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, People’s Republic of ChinaABSTRACT 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.https://journals.asm.org/doi/10.1128/mSystems.00198-19emulsionmetagenomicsmicrofluidic chiprespiratory microbiome |
| spellingShingle | 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 mSystems emulsion metagenomics microfluidic chip respiratory microbiome |
| title | 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_short | 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 |
| topic | emulsion metagenomics microfluidic chip respiratory microbiome |
| url | https://journals.asm.org/doi/10.1128/mSystems.00198-19 |
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