title: GM standard, primers V1-V2M, replicate 2
design: Genomic DNA  was amplified in PCR reaction with primers targeting the variable regions V1-V2 and V4 of the 16S rRNA gene.  Amplification of both variable regions was performed in 50 L reactions, containing 20 L of Platinum II Hot-Start PCR Master Mix (2X) (Thermo Fisher Scientific, Waltham, USA), 0.1-0.3 molL-1 of primers and 6 L template. The SPRIselect beads (Beckman Coulter, California, USA) were used for PCR product purification. The PCR products were pooled at a standardized concentration of 4 nM. The pooled library was prepared and subjected to MiniSeq Mid Output Kit (2 x 150 paired-end sequencing) on the MiniSeq sequencer (Illumina, San Diego, California, USA) using custom sequencing primers for V4 and V1V2 (see Table 1). Raw fastq reads were mapped to the human genome hg38 using Rbowtie2 package (version 1.14.0 ). Successfully mapped reads were then subtracted from the dataset. The rest reads were processed using the DADA2 package (version 1.20.0) in R (version 4.1.1).