Miseq reagent kit v3 600

For library generation the V3 and V4 region of 16S rRNA region was amplified by PCR with 30 cycles from the extracted DNA. PCR protocol, primer, and library generation were performed exactly as described by (Illumina (2013) using MiSeq Reagent Kit v3 600‐cycles (Illumina, San Diego CA., Cat. No. S102‐3003). Data were acquired using the MiSeqDx System MiSeq and metagenomic analysis of the raw data was performed using the in‐system software MiSeq Reporter. For taxonomic classification, the Greengenes Database files were used (Mc Donald et al., 2012). In Greengenes an OTU refers to the terminal level at which the sequence is classified.

Two panels containing targeted gene BRCA1/2 were used for next-generation sequencing (NGS). Genetic analysis of the probands and of a part of their relatives was performed through the enrichment protocol TruSight Cancer (Illumina, San Diego, CA, USA), as previously reported [23 (link)]. A further part of the subjects was analyzed using the v1.1 SOPHiA Hereditary Cancer Solution (HCS) enrichment protocol (SOPHiA GENETICS, Saint-Sulpice, Switzerland) and output files (FASTQ) uploaded on the SOPHiA DDM Platform v5.5.0 (SOPHiA GENETICS, Saint-Sulpice, Switzerland) for the analysis, as already described by our group [24 (link)] Sequencing was performed using the MiSeq sequencer platform (Illumina) and MiSeq Reagent Kit v2 or MiSeq Reagent Kit v3 600 cycles. According to the International Agency for Research on Cancer (IARC) recommendations [25 (link)], BRCA1/2 variants were classified as pathogenic (PV; class 5) and likely pathogenic (LPV; class 4) through the major variants databases and tool prediction software [26 (link)].

The cDNA library was made from purified circRNA solution by using NEBNext Ultra II RNA Library Prep Kit for Illumina E7770 (New England Biolabs, Ipswich, MA, USA) according to the instructions of the manufacturer. The prepared library was quantified by NebNext Library Quantification Kit for Illumina. Fragmentation sizing was carried out by High Sensitivity dsDNA chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Next-generation sequencing was performed by MiSeq Reagent Kit v3 600 on Illumina MiSeq instrument (Illumina, San Diego, CA, USA).

The 16S rRNA gene metabarcoding of 116 out of 120 samples was performed following the protocol suggested by Illumina (four samples were discarded as they were not of adequate quality for sequencing). Briefly, 22.5 ng of cDNA was used as input for the first PCR using 16S amplicon PCR forward and reverse primers, amplifying V3-V4 regions of the 16S rDNA. After purification and second (index) PCR with a Nextera XT Index kit (Illumina, San Diego, CA, USA), the libraries were normalized according to fragment length and dsDNA molarity. The samples were pooled and processed in four sessions on a MiSeq platform (Illumina) using a MiSeq reagent kit v3-600 for 2x300 paired-end sequencing at the IGA Technology Services facility. The datasets generated and analyzed for this study are available in the BioProject database, with SubmissionID: SUB2898018 and BioProject ID: PRJNA396024.

Next generation sequencing was used for whole-genome sequencing of K. pneumoniae isolates before and after the co-culture. The bacterial DNA was extracted from freshly sub-cultured colonies using the QIAamp DNA Mini Kit (Qiagen, cat # 51304) according to the manufacturer’s instructions, and the DNA concentration was measured using a Denovix Fluorometer (Denovix, Wilmington, DC, USA). The genomic DNA was stored at −20 °C. Total bacterial DNA (1 ng) was used in the library preparation. The library was prepared with Nextera XT DNA Library Preparation Kit (FC-131-1096, Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. To summarize, transposons were used to fragment the DNA, subsequently, adapter sequences were added onto the DNA template. The product was then size-selected for optimum insert length, enriched, and quantified. Sequencing was carried out with the MiSeq reagent kit 600 v3 (Illumina, USA) on the Illumina MiSeq, generating, on average, 301 base pair paired-end reads.
FastaQ files were uploaded on Illumine BaseSpace and were assembled de novo using the Spades application. The assembled FASTA contig files were uploaded to both the PATRIC online tool and the Center of genomic epidemiology online website database for further analysis.

Full-length S gene sequencing of wastewater samples was performed
following the ARTIC Network protocol (https://artic.network/ncov-2019,
with minor modifications) with selected v3 primers (Integrated DNA
Technologies) for genome amplification and KAPA HyperPrep Kit (Roche
Applied Science) for library preparation.^{23 (link)} Libraries were loaded in MiSeq Reagent Kit 600v3 cartridges and
sequenced on a MiSeq platform (Illumina). The raw sequenced reads
were cleaned from low-quality segments and mapped against the Wuhan-Hu-1
reference genome sequence to find out variant-specific signature mutations
(mutations and indels).