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| Main Authors: | , , , , |
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| Format: | Artículo científico |
| Language: | en |
| Published: |
ISME communications
2025
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| Online Access: | https://pubmed.ncbi.nlm.nih.gov/41439189/ |
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| _version_ | 1868266108323102720 |
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| author | Ghotbi, Marjan Ghotbi, Mitra D'Agostino, Elisa Kanitz, Maarten Needham, David M |
| author_facet | Ghotbi, Marjan Ghotbi, Mitra D'Agostino, Elisa Kanitz, Maarten Needham, David M Ghotbi, Marjan Ghotbi, Mitra D'Agostino, Elisa Kanitz, Maarten Needham, David M |
| collection | PubMed - marine biology |
| contents | From microscale to microbial insights: validating high-throughput microvolume extraction (HiMEx) methods for marine microbial ecology. Ghotbi, Marjan Ghotbi, Mitra D'Agostino, Elisa Kanitz, Maarten Needham, David M Extracting and directly amplifying DNA from small-volume, low-biomass samples would enable rapid, ultra-high-throughput analyses, facilitating the study of microbial communities where large-volume sample collection is challenging. This can aid where 'conventional' filtrater-based methods miss capturing smaller microbes, or where microscale variability matters, such as the ocean. Here, we develop and validate physical and chemical-based DNA extractions from microvolumes with universal rRNA gene amplicons and metagenomic sequencing of all domains and viruses, on natural surface seawater and experimentally manipulated marine waters. Compared to 500-mL filter-based extraction, direct PCR of 3 μL of lysate from seawater microvolume extractions ranging from 100-1000 μL consistently captured comparable microbial community composition and diversity, with reliable amplification and little to no contamination. Metagenomic results of 10 μL lysates from 15 microvolume samples (100 μL) captured 83 high- and draft-quality, diverse bacterial genomes and 430 complete, high and medium quality viral contigs. Our approach enables scaling of rRNA gene sequencing and metagenomic library prep for high-throughput experimentation for a fraction of the cost of conventional methods and builds upon existing microvolume approaches by removing unnecessary expenses, like excess plasticware and expensive bead clean-up. The method expands opportunities for more comprehensive microbial community monitoring and controlled laboratory experiments by facilitating higher sample numbers and lowering sample volume needs. However, its potential bias against Gram-positive bacteria should be considered when applying to environments where these taxa are abundant. |
| format | Artículo científico |
| id | pubmed_41439189 |
| institution | PubMed |
| language | en |
| publishDate | 2025 |
| publisher | ISME communications |
| record_format | pubmed |
| spellingShingle | From microscale to microbial insights: validating high-throughput microvolume extraction (HiMEx) methods for marine microbial ecology. Ghotbi, Marjan Ghotbi, Mitra D'Agostino, Elisa Kanitz, Maarten Needham, David M From microscale to microbial insights: validating high-throughput microvolume extraction (HiMEx) methods for marine microbial ecology. Ghotbi, Marjan Ghotbi, Mitra D'Agostino, Elisa Kanitz, Maarten Needham, David M Extracting and directly amplifying DNA from small-volume, low-biomass samples would enable rapid, ultra-high-throughput analyses, facilitating the study of microbial communities where large-volume sample collection is challenging. This can aid where 'conventional' filtrater-based methods miss capturing smaller microbes, or where microscale variability matters, such as the ocean. Here, we develop and validate physical and chemical-based DNA extractions from microvolumes with universal rRNA gene amplicons and metagenomic sequencing of all domains and viruses, on natural surface seawater and experimentally manipulated marine waters. Compared to 500-mL filter-based extraction, direct PCR of 3 μL of lysate from seawater microvolume extractions ranging from 100-1000 μL consistently captured comparable microbial community composition and diversity, with reliable amplification and little to no contamination. Metagenomic results of 10 μL lysates from 15 microvolume samples (100 μL) captured 83 high- and draft-quality, diverse bacterial genomes and 430 complete, high and medium quality viral contigs. Our approach enables scaling of rRNA gene sequencing and metagenomic library prep for high-throughput experimentation for a fraction of the cost of conventional methods and builds upon existing microvolume approaches by removing unnecessary expenses, like excess plasticware and expensive bead clean-up. The method expands opportunities for more comprehensive microbial community monitoring and controlled laboratory experiments by facilitating higher sample numbers and lowering sample volume needs. However, its potential bias against Gram-positive bacteria should be considered when applying to environments where these taxa are abundant. |
| title | From microscale to microbial insights: validating high-throughput microvolume extraction (HiMEx) methods for marine microbial ecology. |
| url | https://pubmed.ncbi.nlm.nih.gov/41439189/ |