Miseq analyzer

Whole bacterial DNA from six clinical extraintestinal E. coli was isolated from Luria Broth overnight cultures with use of silica column-based Genomic Mini AX Bacteria kit (A&A Biotechnology). Purified bacterial DNA was sequenced using both short- and long-read methodologies (Illumina and Oxford Nanopore Technology).
In the first step of molecular analysis, Nextera XT library preparation kit and Nextera XT Indexes (Illumina) were used for previously quantified bacterial DNA, which was simultaneously fragmented and tagged with sequencing adapters in a single-tube enzymatic reaction. Quality and quantity of libraries were assessed by fluorometry (Qubit, Thermo Fisher Scientific) and chip electrophoresis (2100 Bioanalyzer, Agilent). FASTQ reads were generated with the use of MiSeq Reagent Kit v3 (600 cycles) and MiSeq analyzer (Illumina).
In the next step, DNA libraries were prepared with the use of a Ligation Sequencing Kit (SQK-LSK109) with Native Barcoding Expansion (EXP-NBD104). Quality and quantity of libraries were assessed by fluorometry (Qubit, Thermo Fisher Scientific) and chip electrophoresis (2100 Bioanalyzer, Agilent). FASTQ reads were generated with the use of Spot-ON Flow Cell (FLO-MIN106D R9 Version) and MinION Mk1b analyzer (Oxford Nanopore Technology).

Hierarchical clustering and ordination of the community structures were performed using a Principal Coordinate Analysis (PCoA) plot by Illumina MiSeq analyzer (Illumina, San Diego, CA, USA). Results are presented as mean ± SEM. The statistical analysis was performed using GrapPad Prism® software v 7.01 (GraphPad Software, Inc., La Jolla, CA, USA). Grubb´s test was performed to determine outliers and the Shapiro-Wilk test was used to check the normality of data distribution. When comparing three groups (i.e. CTL, SDP and COL), the ANOVA test was used when data were normally distributed; otherwise the non-parametric Kruskal-Wallis test was carried out. When only Control and SDP conditions were compared, the Student t-test was used when data were normally distributed; otherwise, the non-parametric Mann-Whitney U-test test was used. All p-values were corrected for multiple testing using the false discovery rate (FDR) correction (Benjamini-Hochberg). Statistical differences were considered significant at q < 0.05. A q value between 0.05 and 0.1 was suggestive of a true effect^{78 (link)}.

Blood samples were collected from each participant for germline DNA testing when the patients were referred to a Genetic Counseling Unit. All participants were screened for BRCA1 and BRCA2 mutations, either by Sanger sequencing using Applied Biosystems’ 3130XL Genetic Analyzer (Thermo Fisher Scientific, Carlsbad, CA, USA) or by massive parallel sequencing using the TruSight Cancer panel (Illumina, San Diego, CA, USA) on a MiSeq Analyzer, as previously described [40 (link)]. Carriers of variants of uncertain/unknown significance (VUSs) were deemed as non-carriers. The NM_007294/ENST00000357654 and NM_000059/ENST00000380152 were used as the reference sequences for BRCA1 and BRCA2 genes, respectively. According to the typical BRCA exon numbering used, the BRCA1 gene has 24 exons and encodes 1863 amino acids, while BRCA2 consists of 27 exons and encodes 3418 amino acids (Figure 4).