Impaired microbiota in breast cancer patients: In association with high lipopolysaccharide levels and a low-fibre diet
For the library preparation of the sample, Ligation sequencing kit (SQK-NBD109.24;Oxford Nanopore Technologies) protocol was used. First the sample was end-prepped,dA tailed and nick-repaired in a final reaction volume of 60 l, then the sample was purified using MobiomX MagBeads and adapters were ligated to the repaired ends. Samples were then purified and quantified spectroflorimetrically. Total library was loaded onto a Spot-On flow cell (FLO-MIN106) after priming and the sequencing run was initiated on an Mk1C device (Oxford Nanopore Technologies) using the latest MinKNOW software. Sequencing was stopped when enough data was obtained or the maximum run time of 72 hours was completed. After sequencing, the results obtained in pod5 format were converted to fastq format using latest version of dorado software (base-calling and de-multiplexing). Barcode and adapter sequences were cleared using dorado software, and universal primers and tags were also deleted by deleting 15 bases from both ends of the sequences. After clearing the sequences, reads 1250-1750 bp long were filtered with Trimmomatic, and the remaining reads were excluded from the analysis. The trimmed reads were aligned to the Massive Bioinformatics database produced using 16S rRNA genes of bacterial whole genomes submitted to NCBI using the Massive Align Engine (MAE). Using the taxonomic details of the 16S rRNA genes to which the reads were aligned and the number of reads aligned to the genes, a biome file was produced. Using the capabilities offered by the qiime2 platform, alpha diversity analysis was carried out using several kinds of indexes in order to conduct phylogenetic analysis using the biome file which had been produced. Utilizing the Mothur platform, taxonomic classifications were arranged. The Python programming language's libraries were used to create the tables and graphs in the analysis.