Qubit 2.0 fluorometer

Extracted DNA was quantified using Qubit 2.0 fluorometer (Invitrogen by ThermoFisher Scientific, Life Technologies Italia, Monza, Italy). Paired-end libraries were prepared from 1 ng of total bacterial DNA using Nextera XT DNA Sample Preparation kit and Nextera XT Index kit (Illumina Inc., San Diego, California, USA) according to manufacturer’s protocol. Library concentration and average fragment size were calculated by Qubit 2.0 fluorometer and Caliper LabChip GXI System (Perkin Elmer, Waltham, USA) respectively. Libraries were then normalized to 2 nM, pooled for multiplexing in equal volumes, and sequenced at 10 pM on the Illumina HiSeq 2000 platform (Illumina Inc., San Diego, California, USA) with 100 nt paired-end reads to achieve a coverage >100x per base.

S. maltophilia isolates were grown on Columbia agar plates with 5% sheep blood (bioMérieux Italia S.p.A, Florence, Italy) for 24 h at 37°C, and genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). DNA was quantified using Qubit 2.0 fluorometer (Invitrogen by Thermo Fisher Scientific, Life Technologies Italia, Monza, Italy). Paired-end libraries were prepared from 1 ng of total bacterial DNA using Nextera XT DNA Sample Preparation kit and Nextera XT Index kit (Illumina Inc., San Diego, California, U.S.A.). Library concentration and average fragment size were calculated by Qubit 2.0 fluorometer and Caliper LabChip GXI System (PerkinElmer, Waltham, USA), respectively. Libraries' concentration was normalized to 2 nM, pooled for multiplexing in equal volumes, and sequenced at 14 pM on the Illumina MiSeq platform (Illumina Inc., San Diego, California, U.S.A.) with 300 nt paired-end reads to achieve a coverage of about 30x per base, using MiSeq V3 flow cell.