CU2.5C
DNA of the samples was amplified using primers targeting the V4 variable region of the bacterial 16SrRNA gene. Primers sequences were: CS1_515F (ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGTAA) and CS2_806R (TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTCTAAT) (Caporaso et al. 2012). These primers contained 5 common sequence tags, in accordance to Moonsamy et al. (2013). The PCR amplification procedure is described in details in Aizenberg-Gershtein et al. (2015). Illumina MiniSeq sequencing was performed at the DNA Services (DNAS) Facility University of Illinois, Chicago (UIC). The sequencing protocol is described in Aizenberg-Gershtein et al. (2015). The procedure included a second PCR amplification in 96-well plates, where each well received a separate primer pair, obtained from the Access Array Barcode Library for Illumina Sequences [10-base barcode (Fluidigm, South San Francisco, CA; Item no. 100-4876)]. The pooled library was purified by an AMPure XP cleanup protocol (0.6X, vol/vol; Agencourt, Beckmann-Coulter) to remove smaller fragments than 300 bp. The pooled libraries, with a 20% phiX spike-in, were loaded onto an Illumina MiniSeq mid-output flow cell (2x153 paired-end reads). Based on the distribution of reads per barcode, the amplicons (before purification) were-pooled to generate a more balanced distribution of reads. The re-pooled library was purified by AMPure XP cleanup, as described above. The re-pooled libraries, with a 20% phiX spike-in, were loaded onto a Miniseq flow cell, and sequenced (2x153 paired-end reads). Fluidigm sequencing primers, targeting the CS1 and CS2 linker regions, were used to initiate sequencing. De-multiplexing of reads was performed on instrument. Library preparation, pooling, and sequencing were performed at the University of Illinois at Chicago Sequencing Core (UICSQC) within the Research Resources Center (RRC).