Only two promiscuous probes were shared between both sets of Tag4 data: ED116 and ED121B (G. vaginalis). Whereas one A. baumannii probe (ED211) was promiscuous in the simulated clinical sample data, two other A. baumannii probes (ED212 and ED213) were promiscuous in the
clinical sample data. What remained for the authentic clinical samples assayed on the Tag4 array were (192 – 17 =) 175 molecular probes representing 37 bacteria. Public genome sequence for L. crispatus and L. jensenii appeared only after the design of all of the other molecular probes . These two genome sequences were derived from short shotgun pyrosequencing reads, which had been assembled into dozens of contigs for each genome. Thus, these two genome sequences were far from ideal for the purpose of designing unique 40-mer Homers. Selinexor Nevertheless, given the importance of L. crispatus and L. jensenii
to the health of the human vagina, we designed molecular probes for these two bacterial DNAs. Presumably, as a direct consequence of the incompleteness of the two genome sequences, the molecular probes for L. crispatus and L. jensenii cross-reacted with each other’s DNA and sometimes with L. see more brevis and L. gasseri DNAs as well. In addition, although the Selleck Entospletinib sequences for the existing molecular probes for L. brevis and L. gasseri were compared to the L. crispatus and L. jensenii genome sequences with only negative results, the L. brevis and L. gasseri probes sometimes reacted with L. crispatus and L. jensenii DNAs in the clinical samples. To avoid confusion, only those Lactobacillus species identified by BigDye-terminator
sequencing Rho appear in the tables. The probes for L. acidophilus, L. delbrueckii, and L. plantarum did not cross-react with other Lactobacillus DNAs. The microarray data are MIAME compliant and have been deposited in the Array Express website: accession: [E-MEXP-2958]. The CEL (cell intensity) files of the microarray data are publicly available on the Stanford Genome Technology Center website http://med.stanford.edu/sgtc/research/download/. Assaying the molecular probes by Sequencing by Oligonucleotide Ligation and Detection (SOLiD) The primers used to amplify the product for SOLiD sequencing are presented in Table S1 (Additional file 1). The primer sequences were based upon published designs . SOLiD sequencing, a sequencing-by-ligation technology (Applied Biosystems, Foster City, CA), was performed at the University of California Santa Cruz Genome Sequencing Center. We have published our procedure for the library preparation of the samples for SOLiD sequencing [25, 26]. We followed the manufacturer’s protocols for the barcoded SOLiD System 3.0 Fragment Library. We prepared the samples according to the manufacturer’s protocols for the emulsion PCR step of SOLiD sequencing. We processed the samples with the SOLiD Version 3.0 system, producing 50 bases of sequencing information for each read.