PoolRosalia
After thawing and vigorously homogenization for 1 min, 1 mL of fecal sample was disrupted by the Lysing Matrix E extraction beads (MPbio) and clarified by centrifugation at 6,000 g for 10 min at 4C. Between 100-250 uL of fecal supernatants or the direct PBS from swab samples were mixed with samples from animals of the same location, resulting in 16 pools (GLT = 7 and Marmoset = 9). Sampling pools are summarized in Table 1. The pooled samples were filtered through an Ultra-free-MC HV 0.45 m sterile filter (Millipore, UFC30HV0S). The filtrates were submitted to sucrose density gradient ultracentrifuge separation at 35,000 g for 90 min at 4C and degradation of unprotected nucleic acid by nuclease digestion at 37C for 60 min. Remaining total nucleic acid were then isolated using QiaAmp MiniViral RNA kit (Qiagen), followed the manufacturers instructions, with the following  modifications: i) the "Carrier RNA" was omitted from the AL Buffer; ii) the protease was resuspended in AVE Buffer, instead of Protease Resuspension Buffer; iii) the washing step with AW1 was suppressed; iv) the final elution was performed in 20 L of ultra-pure water. Thereafter, a RT-PCR reaction was performed with SuperScript III First-Strand Synthesis System (Invitrogen) for first-strand cDNA synthesis from RNA, using random primers, while preserving DNA. The second-strand cDNA synthesis was performed using DNA Polymerase I Large (Klenow 3'-5' exo) Fragment (New England Biolabs). All those reactions were conducted according to the manufacturers instructions. DNA quantification was performed using the High Sensitivity dsDNA Assay kit in a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). The libraries were constructed using the NexteraTM XT  DNA Library Preparation Kit, purified with the Agencourt AMPure XP - PCR Purification (Beckman Coulter) and quantified using both High Sensitivity DNA Kits from Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and 2100 Bioanalyzer (Agilent Technologies). The sequencing was conducted by applying 2pM of each library in the MiSeq Illumina platform using the MiSeq V2 300-cycle kit (Illumina) in paired-end mode 2  150 bp with dual barcode for each pooled sample.