We analyzed Streptococcus Group I (SGI) and Streptococcus Group II (SGII) CRISPRs, by amplifying them based on their consensus repeat motifs (Additional file 1: Table S1) [14, 15]. These CRISPR repeat motifs are present in a variety of different streptococcal species, including S. pyogenes and S. agalactiae that are primarily found on the skin, and numerous different viridans streptococci such as S. mutans, S. gordonii, S. mitis, and S. sanguinis that are found in the oral cavity (Additional file 1: Table S2). The benefits of this approach were that we could analyze CRISPR spacers from numerous streptococcal Evofosfamide order species simultaneously and were not limited to examining individual CRISPR loci.

Ruxolitinib The main drawbacks of this technique were that it was difficult to ascribe the spacers to any single CRISPR locus or bacterial species, and the consensus repeat motifs could be present in some non-streptococcal species. We amplified CRISPRs from all subjects, sample types, and www.selleckchem.com/products/jnk-in-8.html time points, and sequenced 4,090,937 CRISPR spacers consisting of 2,212,912 SGI and 1,878,025 SGII spacers using semiconductor sequencing [36] (Additional file 1: Table S3). There were 2,169,768 spacers obtained from saliva and 1,921,169 spacers obtained from skin. For all time points combined, we

found 1,055,321 spacers for Subject #1, 781,534 spacers for Subject #2, 1,088,339 for Subject #3, and 891,618 spacers for Subject #4. Spacer binning and estimated coverage We binned each of the CRISPR spacers according to trinucleotide content according to our previously described

protocols [10]. The majority of the CRISPR spacers identified in each subject and time point were identical to other spacers, with only 0.001% of SGI and 0.002% of SGII spacers identified as having polymorphisms that necessitated grouping according to trinucleotide content. We sequenced an average of 28,333 spacers per time point and sample type in each subject to capture the majority of the CRISPR spacer diversity in these environments. We then performed rarefaction analysis on all subjects by CRISPR and sample these type to estimate how thoroughly each had been evaluated. We found that all curves neared asymptote for all subjects, sample types, and time points, with the exception of Subject#1 in the evening of week 8 for SGII CRISPR spacers (Additional file 2: Figure S1). CRISPR spacer distribution We compared CRISPR spacers and their relative abundances across all time points in each subject to determine how spacers in each subject were distributed over time. At each time point, many of the spacers found at early time points persisted throughout later time points (Figure 1 and Additional file 2: Figure S2), indicating that many of the SGI and SGII CRISPR spacers were conserved throughout the study period.