Miseq desktop sequencer

Next generation sequencing was performed as described elsewhere (Scholz et al., 2016 (link)). In brief: isolated single eGFP-positive cells were identified, picked and transferred to a PCR tube using a combined confocal laser-scanning/patch-clamp setup (Leica TCS SP5, Leica Microsystems/Luigs-Neumann, Wetzlar/Ratingen, Germany). Cell lysis, cDNA generation and amplification were performed using the Sigma SeqPlex RNA Amplification Kit (Sigma-Aldrich/Merck, Darmstadt, Germany). For library preparation, the Illumina Nextera XT DNA sample preparation protocol (Part # 15031942 Rev. C) was used. Samples run together with a 2 × 75 bp read length using the MiSeq Reagent Kit v3 (150 cycles) and the Illumina MiSeq Desktop Sequencer (Illumina, San Diego, CA, USA). The sequencing reads were aligned to the mm9 reference genome and transcriptome using TopHat2 (2.0.9). The TopHat output files were saved in BAM format and evaluated by Cuffdiff2 (2.1.1). All samples were compared and evaluated in one calculation cycle, allowing the algorithm to estimate the Fragments Per Kilobase Million (FPKM) values at the transcript level resolution and to control for variability across the replicate libraries.

For total DNA isolation, frozen samples were thawed, treated with DTT (50 µg/ml at 37°C for 20 min) and 250 µl of the sample was used for isolation according to published protocols in a MagnaPure LC device (Roche, Mannheim, Germany) with the MagnaPure LC DNA III Isolation Kit (Bacteria, Fungi) (Roche, Mannheim, Germany) (28 (link)). DNA was eluted in 50-µl elution buffer (Roche, Mannheim, Germany) and stored at -20°C till analysis. For 16S rRNA gene PCR, 2 µl of template DNA were used in a standard PCR setup in 25 µl reactions in triplicates with Roche High Fidelity Polymerase (Mannheim, Germany) and the target-specific primers 515F (GTGYCAGCMGCCGCGGTAA) and 926R (CCGYCAATTYMTTTRAGTTT), 30 amplification cycles, according to published protocols (28 (link)). The indexing of samples, pooling, purification, and quality control were performed as published elsewhere, and the final library was sequenced at 9pM with 20% PhiX on an Illumina MiSeq desktop sequencer (Illumina, Eindhoven, Netherlands) with v3 600 cycles chemistry in 2×300 sequencing mode. FASTQ files were used for data analysis, and raw data were archived in the European Nucleotide Archive (ENA) and can be downloaded with the accession number PRJEB51689 (https://www.ebi.ac.uk/ena/; last access March 2022).

Once the concentration of the extracted double-stranded DNA had been measured using the Quant-iT^TM Picrogreen® kit (Invitrogen, Inc., Carlsbad, USA), it was amplified with a Nextera® transposase sequences (details at S11 Text in supplementary information) and KAPA HiFi^TM PCR kit (Roche life science, Inc., Indiana, USA). The PCR products were then purified by Agencourt® AMPure XP beads (Beckman Coulter, Inc., Fullerton, USA). In the index PCR, the reaction mixture was attached with the Illumina indexes (Illumina, Inc., San Diego, USA) using the KAPA HiFi^TM PCR kit The DNA libraries produced were purified again using the Agencourt® AMPure XP beads. After the quantity of each library had been checked, it was normalised and pooled as Pooled Amplicon Library (PAL). Then, the PAL was then re-quantified with qPCR using the KAPA Library Quantification Kit (Roche life science, Inc., Indiana, USA).
The PAL and the internal control library (Illumina, Inc., San Diego, USA) were denatured with sodium hydroxide (NaOH) and diluted with a pre-chilled hybridization buffer (HT1) to the appropriate final concentration. The solution was sequenced on the Illumina® Miseq Desktop Sequencer (Illumina, Inc., San Diego, USA) The detailed protocol of the 16S rRNA sequencing preparation was described in Supplementary Text 11.

Genomic DNA was extracted after overnight incubation at 35 °C in NB using the DNeasy Blood and Tissue Kit (Qiagen Inc, Valencia, CA). DNA concentrations were measured using a Qubit 3.0 fluorometer (Life Technologies, MD). Sequencing libraries were prepared according to Nextera XT protocols using 0.2 ng/μl of DNA and sequenced on the Illumina MiSeq desktop sequencer (Illumina, San Diego, CA) using MiSeq Reagent V2 kits (500 cycles of paired end reads) following the manufacturer’s guidelines. Sequences were trimmed using the Illumina software using standard parameters. The resulting trimmed Fastq data sets were de novo assembled using Unicycler v0.4.8 with default parameters. Assembled genomic data was submitted to PATRIC and Rapid Annotation using Subsystem Technology tool kit (RASTtk) [20 (link), 21 (link)] for annotation and comparative analysis, to predict the presence of genes relevant to risk assessment (virulence factors, antibiotic resistance genes, drug targets, and human homologs).