PreprintArticleVersion 1This version is not peer-reviewed
Benchmarking of DNA Extraction Methods for Metagenomics, 16S rRNA Databases and Assessment of the Skimmed Milk Flocculation for Bacterial Enrichment for Metagenomics from Water Samples
Version 1
: Received: 9 September 2024 / Approved: 10 September 2024 / Online: 10 September 2024 (10:08:12 CEST)
How to cite:
Donchev, D.; Stoikov, I.; Diukendjieva, A.; Ivanov, I. N. Benchmarking of DNA Extraction Methods for Metagenomics, 16S rRNA Databases and Assessment of the Skimmed Milk Flocculation for Bacterial Enrichment for Metagenomics from Water Samples. Preprints2024, 2024090722. https://doi.org/10.20944/preprints202409.0722.v1
Donchev, D.; Stoikov, I.; Diukendjieva, A.; Ivanov, I. N. Benchmarking of DNA Extraction Methods for Metagenomics, 16S rRNA Databases and Assessment of the Skimmed Milk Flocculation for Bacterial Enrichment for Metagenomics from Water Samples. Preprints 2024, 2024090722. https://doi.org/10.20944/preprints202409.0722.v1
Donchev, D.; Stoikov, I.; Diukendjieva, A.; Ivanov, I. N. Benchmarking of DNA Extraction Methods for Metagenomics, 16S rRNA Databases and Assessment of the Skimmed Milk Flocculation for Bacterial Enrichment for Metagenomics from Water Samples. Preprints2024, 2024090722. https://doi.org/10.20944/preprints202409.0722.v1
APA Style
Donchev, D., Stoikov, I., Diukendjieva, A., & Ivanov, I. N. (2024). Benchmarking of DNA Extraction Methods for Metagenomics, 16S rRNA Databases and Assessment of the Skimmed Milk Flocculation for Bacterial Enrichment for Metagenomics from Water Samples. Preprints. https://doi.org/10.20944/preprints202409.0722.v1
Chicago/Turabian Style
Donchev, D., Antonia Diukendjieva and Ivan N. Ivanov. 2024 "Benchmarking of DNA Extraction Methods for Metagenomics, 16S rRNA Databases and Assessment of the Skimmed Milk Flocculation for Bacterial Enrichment for Metagenomics from Water Samples" Preprints. https://doi.org/10.20944/preprints202409.0722.v1
Abstract
Water samples for bacterial microbiome studies undergo a biomass concentration, DNA extraction and taxonomic identification steps. Through benchmarking, we studied the applicability of Skimmed milk flocculation (SMF) for bacterial enrichment, an adapted in-house DNA extraction protocol, and six 16S rRNA databases (16S-DBs). Surface water samples from two rivers were treated with SMF and Vacuum filtration (VF) and subjected to amplicon or shotgun metagenomics. A mock community standard underwent five DNA extraction protocols, taxonomical identification with six different 16S-DBs and evaluated by the Measurement Integrity Quotient (MIQ) score. In SMF samples, the skimmed milk was metabolized by members of lactic acid bacteria or genera such as Polaromonas, Macrococcus, and Agitococcus, resulting in increased relative abundance (p < 0.5) up to 5.0 log fold change compared to VF, rendering SMF inapplicable for bacterial microbiome studies. The best performing DNA extraction protocols were FastSpin Soil, the in-house method and EurX. All 16S-DBs yielded comparable MIQ scores within each DNA extraction kit, ranging from 61-66 (ZymoBIOMICs) up to 80-82 (FastSpin). DNA extraction kits exert more bias to the composition than the 16S-DBs. This benchmarking study presented valuable insights to inform future water metagenomic study designs.
Keywords
skimmed milk flocculation; vacuum filtration; bacterial metagenomics; benchmarking; DNA extraction; water samples; taxonomic composition; MIQ score; mock standard
Subject
Biology and Life Sciences, Immunology and Microbiology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
