Spri te instrument

Bacterial DNA was purified from FFPE tissue sections using a commercial kit (NucleoSpin Kit, Macherey-Nagel, Düren, Germany) as recently described [9 (link), 10 (link)]. A total of 8100 ng of total DNA was extracted and used for NGS. Purified DNA was fragmented by sonication (M220 Focused-Ultrasonicator; Covaris, Woburn, Massachusetts, USA) and 500 ng of the fragmented DNA was used as input for library preparation with the aid of a SPRI-TE instrument (Beckman Coulter, Krefeld, Germany) with SPRIworks II cartridges and NEXTflex-96 DNA Barcodes (Bioo Scientific, Austin, TX, USA). Library preparation was done without automatic size selection and the resultant libraries were instead manually size selected (peak size 500 bp) with Ampure XP Beads (Beckman Coulter). Finally, the size selected libraries were quantified using the KAPA Library Quantification Kit, Illumina/Universal (KAPA Biosystems, Cape Town, South Africa) and sequenced with an Illumina MiSeq instrument (MiSeq Reagent Kit v2 (500 cycle); Illumina, San Diego, USA). The raw reads were analyzed using RIEMS (zit).
To clarify relevant results of DNA sequences and associated bacterial families, a deliberate mark of all families with a quantity of >40 reads was set and these families were selected (Table 1).

DNA extractions were done using the Puregene Tissue Kit from Qiagen® following the manufacturer's instructions. Then, 500 ng of DNA were sheared to a 150–700 bp range using the Covaris® E210 instrument (Covaris, Inc., USA). Sheared DNA was used for Illumina® library preparation by a semi-automatized protocol. Briefly, end repair, A tailing and Illumina® compatible adaptors (BiooScientific) ligation were performed using the SPRIWorks Library Preparation System and SPRI TE instrument (Beckmann Coulter), according to the manufacturer protocol. A 300–600 bp size selection was applied in order to recover the most of fragments. DNA fragments were amplified by 12 cycles PCR using Platinum Pfx Taq Polymerase Kit (Life® Technologies) and Illumina® adapter-specific primers. Libraries were purified with 0.8× AMPure XP beads (Beckmann Coulter). After library profile analysis by Agilent 2100 Bioanalyzer (Agilent® Technologies, USA) and qPCR quantification, the libraries were sequenced using 100 base-length read chemistry in paired-end flow cell on the Illumina HiSeq2000 (Illumina®, USA).