16S rRNA Gene Amplicon Sequencing of Microbial Communities

DNA was extracted from early enrichments at 37, 50, and 70°C using the MO Bio PowerSoil DNA isolation kit (Qiagen). 16S rRNA gene amplicon libraries were prepared according to the Illumina 16S metagenomic sequencing library preparation protocol (support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). We amplified the V3–V4 region for bacteria and the V4–V6 region for archaea (Supplementary Table S1). The 16S rRNA gene libraries were sequenced at CeBiTec (Bielefeld, Germany) on a MiSeq (Illumina; 2 × 300-bp paired-end run, 100 000 reads per library). Sequences were analyzed in R Statistical Software v 3.5.1 (R-project.org/) with DADA2 v. 1.14 [41 (link)]. DADA2 scripts used for 16S rRNA gene analysis are accessible on GitHub (github.com/dbenitom/Metagenomics_scripts/blob/main/dada2_archaea.R and github.com/dbenitom/Metagenomics_scripts/blob/main/dada2_bacteria.R).

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Benito Merino D., Lipp J.S., Borrel G., Boetius A, & Wegener G. (2024). Anaerobic hexadecane degradation by a thermophilic Hadarchaeon from Guaymas Basin. The ISME Journal, 18(1), wrad004.

Publication 2024

MO Bio PowerSoil DNA isolation kit 16S metagenomic sequencing library preparation protocol

Corresponding Organization : University of Bremen

Other organizations : Institut Pasteur, Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und Meeresforschung

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Variable analysis

independent variables

Incubation temperature (37°C, 50°C, 70°C)

dependent variables

Taxonomic composition of microbial communities based on 16S rRNA gene amplicon sequencing

control variables

DNA extraction method (MO Bio PowerSoil DNA isolation kit)
16S rRNA gene regions sequenced (V3-V4 for bacteria, V4-V6 for archaea)
Sequencing platform (Illumina MiSeq, 2 x 300-bp paired-end run)
Sequencing depth (100,000 reads per library)
Bioinformatics analysis pipeline (DADA2 in R Statistical Software)

controls

Positive control: None mentioned
Negative control: None mentioned

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