In-depth genomic analysis of Granulicatella adiacens causing infective endocarditis
DNA extraction was carried out following the manufacturer instructions provided by QIAGEN QIAamp DNA Mini Kit (Ref. 51304, QIAGEN, 40724 Hilden, Germany). DNA was quantified using both the Eppendorf BioPhotometer D30 and the fluorimeter Qubit dsDNA BR Assay Kit (Ref. 32850, Invitrogen, 92008 Carlsbad, CA, USA). A concentration of 10 ng of each sample was used for NGS sequencing by Illumina MiSeq platform. Libraries were prepared using a standard Illumina library preparation protocol (Nextera XT DNA Library Prep Kit) optimized for bacterial whole-genome sequencing. Genomic DNA was initially fragmented by Nextera XT kit, which simultaneously fragments the DNA and tags the fragments with specific adapter sequences. Sequencing adapters containing the P5 and P7 sequences, as well as unique dual indices (barcodes), were ligated to the DNA fragments. Unligated adapters and small fragments were subsequently removed via magnetic bead purification (Size Selection). The resulting fragments were amplified using Polymerase Chain Reaction (PCR) to enrich the library and add the full sequencing adapter sequences. Libraries were quantified and their quality evaluated using the fluorometric Qubit dsDNA HS Assay Kit (Ref. Q32851, Invitrogen, Carlsbad, CA 92008, USA) to ensure a final average insert size in the range of 300-600 bp. Finally, sequencing was performed by the MiSeq Reagent Kits v3 (Ref. 15043895, Illumina, Inc., 92122, San Diego, CA, USA), using a Paired-End (2x250 bp) run, generating 250 bases from both ends of the DNA fragment. The Sample Sheet was created using the Local Run Manager v3 software and following the instructions in the Local Run Manager v3 Software Guide provided by Illumina.