In brief, we achieved four 96-well plates of sequence reads per swab . We assembled the individual sequence reads into contigs employing the KB Basecaller . Importantly, we hand edited the contigs. We compared the consensus sequence of each contig to the data in the Ribosomal Database Project [RDP; . Technically, the annealing of a molecular probe to a template only confirmed the presence
of a particular sequence. We inferred the presence selleck products of a particular bacterium from the similarity of any given contig consensus sequence to its closest match in the RDP. Molecular probes We have published the detailed design of our molecular probes . In brief, there are three domains within the molecular probes (Figure 1a). The first domain is a contiguous 40-base sequence (the “”Homer”"), divided into two 20-mers, unique to the genome of the target bacteria. A list of the bacteria and their corresponding genome sequences CUDC-907 supplier is provided in (Additional file 1: Table S3) . The second domain is a twenty base oligonucleotide barcode from the Affymetrix Tag4 array . The third domain is a 36-base universal PCR amplification sequence . Thus, the molecular probes are 96 bases in length. We purchased the probes as 5′-phosphorylated
and PAGE-purified from Integrated DNA learn more Technologies. The molecular probe mixture contained 192 molecular probes representing 40 bacteria . There was an average of (192/40 =) 4.8 molecular probes per bacterial genome with a range of 2-to-7. Our procedure is to anneal the molecular probes to the denatured DNA target. Where Pregnenolone there is sufficient sequence similarity between probe and target, a circular DNA forms (Figure 1b). No bases are missing. Only a phosphodiester bond is missing between the 5′ and 3′ bases of the probe.
Enzymatic ligation produces single-stranded circular DNA. Exonuclease digestion removes all linear DNA. PCR primers based upon the 36-base universal amplification sequence are employed to PCR amplify the circular DNA. For the purposes of this work, we excluded from the analysis those bacteria with insufficient public genome sequence to design molecular probes. This category included novel bacteria, which were defined as previously . The novel rDNA sequences have been deposited in GenBank: accession numbers [HQ293151-HQ293203]. Assaying the molecular probes on Tag4 arrays The Tag4 array contains 8-μm features. Each 20-mer barcode is replicated and dispersed five times on the array . We have published the detailed procedures for assaying the molecular probes on the Tag4 array . In all cases, the final read-out was fluorescence intensity. On all the Tag4 arrays, the six molecular probes for L. delbrueckii produced no signals above background (unoccupied 20-mers on the Tag4 array). Therefore, we employed these six probes as the negative controls. We calculated the average fluorescence signal and standard deviation for the six L.