ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples
Abstract Background One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard p...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-12-01
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| Series: | BMC Microbiology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12866-021-02391-z |
| _version_ | 1819009234779504640 |
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| author | Isabel Abellan-Schneyder Andrea Janina Schusser Klaus Neuhaus |
| author_facet | Isabel Abellan-Schneyder Andrea Janina Schusser Klaus Neuhaus |
| author_sort | Isabel Abellan-Schneyder |
| collection | DOAJ |
| description | Abstract Background One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols. Results Using a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts. Conclusion Our improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared. |
| first_indexed | 2024-12-21T00:53:08Z |
| format | Article |
| id | doaj.art-06df479deb10444a9714f74b4607114e |
| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-12-21T00:53:08Z |
| publishDate | 2021-12-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj.art-06df479deb10444a9714f74b4607114e2022-12-21T19:21:21ZengBMCBMC Microbiology1471-21802021-12-0121111410.1186/s12866-021-02391-zddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samplesIsabel Abellan-Schneyder0Andrea Janina Schusser1Klaus Neuhaus2Core Facility Microbiome, ZIEL – Institute for Food & Health, Technische Universität MünchenCore Facility Microbiome, ZIEL – Institute for Food & Health, Technische Universität MünchenCore Facility Microbiome, ZIEL – Institute for Food & Health, Technische Universität MünchenAbstract Background One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols. Results Using a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts. Conclusion Our improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.https://doi.org/10.1186/s12866-021-02391-zddPCRVery small DNA amountsLow-biomass samples16S rRNA gene sequencing |
| spellingShingle | Isabel Abellan-Schneyder Andrea Janina Schusser Klaus Neuhaus ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples BMC Microbiology ddPCR Very small DNA amounts Low-biomass samples 16S rRNA gene sequencing |
| title | ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples |
| title_full | ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples |
| title_fullStr | ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples |
| title_full_unstemmed | ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples |
| title_short | ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples |
| title_sort | ddpcr allows 16s rrna gene amplicon sequencing of very small dna amounts from low biomass samples |
| topic | ddPCR Very small DNA amounts Low-biomass samples 16S rRNA gene sequencing |
| url | https://doi.org/10.1186/s12866-021-02391-z |
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