16s Rrna seq of gut microbiota in stool sample
Stool samples were obtained patients before initiation of therapy and from patients after three to five cycles during therapy using a stool collection tube with 8ml DNA stabilization Buffer  and frozen aliquots were preserved at -80C until further processing. bead-beating and heat-treatment were used for cell lysis and gDNA columns were employed to purify metagenomic DNA. The V3/V4 region of 16 S ribosomal RNA (rRNA) genes was amplifed (25 cycles) from 24 ng DNA using primers 341F and 785R49. After purification using the AMPure XP system (Beckmann Coulter Biomedical GmbH), sequencing was carried out in paired-end mode (PE275) with pooled samples using a MiSeq system (Illumina, Inc., San Diego, California, USA) following the manufacturers instructions and a final DNA concentration of 10 pM and 15% (v/v) PhiX standard library. The generated raw read files were pre-processed using the IMNGS platform (19), a pipeline based on the UPARSE approach (20)  to build sample-specific sequence databases and OTU-based profiles. We then further analyzed generated data using the Rhea pipeline in R studio