Phix control v3 library

The genomic DNA from the stool samples was extracted using a ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. The quality and quantity of the DNA samples were evaluated by Implen NanoPhotometr N60 UV-Vis spectrophotometry (Implen NanoPhotometer, Los Angeles, CA, USA).
Bacterial DNA sequences were evaluated using amplicon sequencing with universal primers 515F and 806R, targeting the V3-V4 hypervariable regions of the 16S rRNA gene. The library preparation was performed according to the 16S Metagenomic Sequencing Library Preparation—Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System protocol in Illumina technology [55 ]. The paired-end sequencing (2 × 300 bp) was performed using the MiSeq platform (Illumina, San Diego, CA, USA) at the Medical University of Warsaw, Warsaw, Poland. For each sequencing run, each 16S rRNA amplicon pool was spiked-in with 10% of the reference PhiX Control v3 Library (Illumina) for improvement of the overall run quality. The sequencing run was performed with 10% of the reference PhiX Control v3 Library (Illumina) spike-in to improve the sequencing quality of 16S rRNA amplicon low-diversity libraries.

All steps were performed according to the 16S Metagenomic Sequencing Library Preparation—Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System protocol (Illumina). Briefly, V3 and V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using 515F and 806R primers. Libraries were prepared with Nextera XT Index Kit (Illumina) followed by paired-end sequencing (2×300 bp) using MiSeq System (Illumina, San Diego, CA, United States). The sequencing run was performed with 10% of reference PhiX Control v3 Library (Illumina) spike-in to improve sequencing quality of 16S rRNA amplicon low diversity libraries. The sequencing was performed at the Medical University of Warsaw in Poland.