16s metagenomic sequencing library preparation protocol

Total DNA was extracted from frozen feces samples using the PowerFecal DNA isolation kit (MOBIO, Carlsbad, CA), following the instructions of the manufacturer. However, a sonication step was introduced before vortexing the bead tubes with fecal samples horizontally for 15 minutes, as described by Jackson and colleagues [15 (link)]. PCR amplification and 50 Amplicon sequencing were performed, using the primer pairs 341F and 806R spanning the V3-V4 hypervariable regions of the 16S rRNA gene along with Illumina adapters, and the Illumina 16S metagenomic sequencing library preparation protocol (15,044,223 Rev. A), respectively. The sequences were de-multiplexed based on the dual index sequences, using the Miseq built-in metagenomics workflow to develop FASTQ files. Data are available upon request.

The metagenomic workflow was carried out according to Bona et al. [31 (link)]. Briefly, total genomic DNA was extracted by DNeasy^® PowerSoil^® Kit (Qiagen, Hilden, Germany) using 0.25 g of soil following the manufacturer’s instructions. DNA of each sample was quantified by fluorimetric method according to the Qubit^® 2.0 Fluorimeter protocol.
Libraries were prepared by following Illumina 16S Metagenomic sequencing library preparation protocol in two amplification steps: an initial PCR amplification using locus-specific PCR primers and a subsequent amplification that integrates relevant flow-cell binding domains and unique indices (NexteraXT Index Kit, FC-131-1001/FC-131-1002). Primer sequences were: 16S-341F 5′-CCTACGGGNBGCASCAG-3′; 16S-805R 5′-GACTACNVGGGTATCTAATCC-3′, amplifying the hypervariable V3-V4 regions. Libraries were sequenced on NovaSeq instrument (Illumina, San Diego, CA, USA) using 250-bp paired-end mode.

Concentrated cells from the CellTraps were centrifuged at 21 000 × g for 30 min prior to being re-suspended in 180 μL ATL buffer (Qiagen) and 20 μL proteinase K (20 mg mL⁻¹). The filters were removed from the falcon tubes, placed in 2 mL eppendorfs with 540 μL ATL buffer (Qiagen) and 60 μL proteinase K (20 mg mL⁻¹) added to them. Sample tubes from both methods were incubated at 55 °C for 30 min and DNA extraction and PCR amplification followed the same procedure as described in Pearman et al.^{16 (link)}. The primers used for amplification targeted the v3 and v4 regions of the 16S rRNA gene^{96 (link)} and the v4 region of the 18S rRNA gene^{97 (link)}. Subsequent to the PCR amplification (where a no negative control was also run) samples were cleaned and normalized using a SeqPrep Normalization plate prior to MiSeq library preparation. The library was prepared following the Illumina 16S metagenomic sequencing library preparation protocol. Before sequencing the samples were cleaned and normalized a second time and tagged samples were pooled for sequencing on a MiSeq sequencing platform at the King Abdullah University Core Laboratory. Raw reads were submitted to the NCBI SRA archive and can be accessed under the project accessions: SRP060785 and SRP081162.