Tapestation 4100

Illumina MiSeq 16S libraries were generated following the standard protocol “16S Metagenomic Sequencing Library Preparation, Part # 15044223 Rev B.” Amplicon PCR was performed using PCR primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) with Illumina library adaptors, for 25 cycles of amplification. DNA extraction negative controls (Bianco_1EXT, Bianco_2EXT) and amplicon PCR negative controls were included in library preparation. Libraries quantification through TapeStation 4100 (Agilent) showed no amplicon signal for amplicon PCR negative controls, which were excluded from the sequencing. In the case of Ae. albopictus samples, amplicon sequencing was run by Macrogen, Inc. using an Illumina MiSeq platform and the Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2. Water samples were sequenced by the Center for Translational Genomics and Bioinformatics – San Raffaele Scientific Institute (Milan, Italy) with Illumina MiSeq 2 × 300 paired-end chemistry (MiSeq Reagent Kit v3).

TruSight Myeloid Panel (TSMP) (Illumina, San Diego, CA, USA), consists of 568 amplicons of 250 base pairs (bp) in length, with a total genomic footprint of 141 kb, targeting the full CDS of 15 genes and exonic hot spots of 39 additional genes (Fig 2) (S1 Table).
Libraries of 17 patient’s samples were prepared by our team following manufacturer’s instructions. Libraries quality was assessed using DNA D1000 kit and a Tape Station 4100 (Agilent Technologies, Santa Clara, CA, USA), and libraries quantity was assessed with Qubit dsDNA HS Assay Kit and Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Libraries were normalized according to the measured quantity and pooled together at 4nM.
A total of 10.5 pM of the 8 pooled libraries was pair-end sequenced on a MiSeq (Illumina, San Diego, CA, USA) with 201x2 cycles using the Reagent Kit V3 600 cycles cartridge, according to manufacturer’s instructions. Bam and Variant Calling Files (VCF) were directly obtained from MiSeq instrument and variants were annotated using Variant Studio (Illumina, San Diego, CA, USA).