title: Fecal microbiome of sarcopenia patient S056
design: The stool DNA was extracted with Mobio PowerFecal DNA Isolation Kit and quantified by using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A two-step polymerase chain reaction (PCR) workflow was performed for library preparation in accordance with procedures described in the Illumina 16S sample preparation guide. The V3-V4 region of the 16S rRNA gene was amplified using a primer overhanging adapter (forward = 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 and reverse = 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3). Dual indices and Illumina sequencing adapters were attached through PCR by using a Nextera XT Index Kit according to the manufacturers instructions. After each PCR process, PCR cleanup was performed using AMPure XP beads to purify the V3-V4 amplicon from the free primer and primer dimer. The sizes of PCR products were verified using the Bioanalyzer DNA 1000 chip. Library quantification was conducted for quality control before sequencing by using the Agilent Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The pooled libraries were then sequenced on the Illumina Miseq platform (Illumina, San Diego, CA, USA) with v3 reagents for paired-end sequencing (2  300 bps) The sequencing workflow has been published previously.