title: AF_category
design: ST..03 Amnionic Fluid  ST..06 Amnionic Fluid  ST..08 Amnionic Fluid  ST..13 Amnionic Fluid  ST..19 Amnionic Fluid  ST..22 Amnionic Fluid  ST..26 Amnionic Fluid  ST..30 Amnionic Fluid  ST..34 Amnionic Fluid  ST..38 Amnionic Fluid  ST..42 Amnionic Fluid  ST..46 Amnionic Fluid  ST..09 inervenous  ST..14 inervenous  ST..16 inervenous  ST..18 inervenous  ST..25 inervenous  ST..29 inervenous  ST..33 inervenous  ST..37 inervenous  ST..41 inervenous  ST..45 inervenous    The V3 and V4 regions of the 16S rRNA gene were amplified using primers compatible with the Nextera Index Kit (Illumina) (NXt_V3-V4_F 5’-TCGTCGGCAGC GTCAGATGTGTATAAG AGACAGCCTAYGGGRB GCASCAG-3’ and NXt_V3-V4_R 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNNGGGTATCTAAT-3’; adapters in bold). PCR reactions containing 12 μl AccuPrimeTM SuperMix II (Life Technologies, CA, USA), 0.5 μl of each primer (10 μM), 5 μl of genomic DNA (~10 ng/ul), and nuclease-free water to a total volume of 20 µl were run on a SureCycler 8800 (Agilent, CA, USA). Cycling conditions applied were: Denaturation at 95°C for 2 min; 35 cycles of 95°C for 15 s, 55°C for 15s and 68°C for 40 s; followed by final elongation at 68°C for 5 min. To incorporate primers with adapters and indexes, PCR reactions contained 12 μl Phusion High-Fidelity PCR Master Mix (Thermo Fisher Scientific, USA, MA), 2 μl corresponding P5 and P7 primer (Nextera Index Kit), 2μl PCR product and nuclease-free water for a total volume of 25 μl. Cycling conditions applied were: 98°C for 1 min; 12 cycles of 98°C for 10 s, 55°C for 20 s and 72°C for 20 s; elongation at 72°C for 5 min. The amplified fragments with adapters and tags were purified using AMPure XP beads (Beckman Coulter Genomic, CA, USA). Prior to library pooling clean constructs were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) and mixed in approximately equal concentrations to ensure even representation of reads per sample followed 250 bp pair-ended MiSeq (Illumina) sequencing performed according to the instructions of the manufacturer.