Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics
Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary ste...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-02-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/5/4270 |
| _version_ | 1797615258299269120 |
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| author | Morgan S. Sobol Anne-Kristin Kaster |
| author_facet | Morgan S. Sobol Anne-Kristin Kaster |
| author_sort | Morgan S. Sobol |
| collection | DOAJ |
| description | Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment. |
| first_indexed | 2024-03-11T07:23:44Z |
| format | Article |
| id | doaj.art-93490272784a4ac7a4f70e492e936b17 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T07:23:44Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-93490272784a4ac7a4f70e492e936b172023-11-17T07:46:15ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-02-01245427010.3390/ijms24054270Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell GenomicsMorgan S. Sobol0Anne-Kristin Kaster1Institute for Biological Interfaces 5 (IBG-5), Karlsruhe Institute of Technology (KIT), 76344 Eggenstein-Leopoldshafen, GermanyInstitute for Biological Interfaces 5 (IBG-5), Karlsruhe Institute of Technology (KIT), 76344 Eggenstein-Leopoldshafen, GermanyMicrobial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment.https://www.mdpi.com/1422-0067/24/5/4270whole genome amplificationminiaturizationcell sortingmicrobial dark mattercontact-free liquid dispenser |
| spellingShingle | Morgan S. Sobol Anne-Kristin Kaster Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics International Journal of Molecular Sciences whole genome amplification miniaturization cell sorting microbial dark matter contact-free liquid dispenser |
| title | Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics |
| title_full | Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics |
| title_fullStr | Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics |
| title_full_unstemmed | Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics |
| title_short | Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics |
| title_sort | back to basics a simplified improvement to multiple displacement amplification for microbial single cell genomics |
| topic | whole genome amplification miniaturization cell sorting microbial dark matter contact-free liquid dispenser |
| url | https://www.mdpi.com/1422-0067/24/5/4270 |
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