Aquagenomic kit

Bacterial DNA was extracted from 15 mg samples using the AquaGenomic Kit (MoBiTec Gmbh, Göttingen, Germany) and further purified using KAPA PureBeads (Roche. Basel. Switzerland) according to the manufacturer’s protocols. The concentration of genomic DNA was measured using a Qubit 3.0 Fluorometer with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Bacterial DNA was amplified with tagged primers (forward 5′TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and reverse 5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) covering the V3–V4 region of the bacterial 16S rRNA gene [19 (link)]. Polymerase chain reactions (PCR) and DNA purifications were performed according to Illumina’s demonstrated protocol (Illumina Inc., San Diego, CA, USA, 2013). The PCR product libraries were quantified and qualified by using High Sensitivity D1000 ScreenTape on TapeStation 2200 instrument (Agilent Technologies, Santa Clara, CA, USA). Equimolar concentrations of libraries were pooled and sequenced on an Illumina MiSeq platform using a MiSeq Reagent Kit v3 (600 cycle; Illumina Inc., San Diego, CA, USA) 300-bp read length paired-end protocol. Raw sequence data of 16S rRNA metagenomics analysis were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive, under the BioProject identifier PRJNA723698.

The extraction of the bacterial DNA was carried out using the AquaGenomic Kit (MoBiTec GmbH, Göttingen, Germany) and further purified using KAPA PureBeads (Roche, Basel, Switzerland) according to the manufacturer’s protocols [14 ]. The genomic DNA was investigated using a Qubit 3.0 Fluorometer with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Bacterial DNA was amplified with tagged primers covering the V3–V4 region of the bacterial 16S rRNA gene [15 (link)]. Polymerase chain reactions (PCR) and DNA purifications were performed according to Illumina’s demonstrated protocol (Illumina Inc., San Diego, CA, USA, 2013). PCR product libraries were defined and qualified using High Sensitivity D1000 ScreenTape on TapeStation 2200 instrument (Agilent Technologies, Santa Clara, CA, USA). Equimolar concentrations of libraries were pooled and sequenced on an Illumina MiSeq platform using a MiSeq Reagent Kit v3 (600 cycle; Illumina Inc.) 300-bp read length paired-end protocol. Raw sequence data of 16S rRNA metagenomics analysis were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under the BioProject identifier PRJNA996958.

Bacterial DNA was extracted from 15 mg samples using the AquaGenomic Kit (MoBiTec GmbH, Göttingen, Germany) and further purified using KAPA Pure Beads (Roche, Basel, Switzerland) according to the manufacturer’s protocols. The concentration of genomic DNA was measured using a Qubit 3.0 Fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Bacterial DNA was amplified with tagged primers (forward, 5′TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, and reverse, 5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) covering the V3–V4 region of the bacterial 16S rRNA gene [18 (link)]. Polymerase chain reactions (PCRs) and DNA purifications were performed according to Illumina’s demonstrated protocol (Illumina Inc., 2013). The PCR product libraries were quantified and qualified by using the High Sensitivity D1000 ScreenTape on the TapeStation 2200 instrument (Agilent Technologies, Santa Clara, CA, USA). Equimolar concentrations of libraries were pooled and sequenced on an Illumina MiSeq platform using a MiSeq Reagent Kit v3 (600 cycle; Illumina Inc., San Diego, CA, USA) with a 300-bp read length paired-end protocol. Raw sequences data of 16S rRNA gene analysis were deposited at the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under accession number PRJNA609272.