Schematic representation of NGS-non-dependent microbiome analysis (NNMA). (a) A sample containing a complex mixture of microbes was serially diluted to a concentration corresponding to one or fewer cell/well as expectation value (in Poisson distribution). DNA extraction and random PCR were performed on the same MMV chip. The PCR product was scaled up using PCR in a 96-well plate and analyzed using μTGGE. In the genome profile obtained, feature points were assigned and processed using computer-aided normalization, generating species identification dots (spiddos). Based on spiddos, a genome distance, d
[defined as 1 − PaSS (pattern similarity score)], was obtained for each pair of microbes (Additional file 12), and a clustering tree was then generated using d
. (b) Genome profiles of four samples with feature points assigned. Red dots represent feature points (pre-spiddos) and yellow dots, internal reference points. Arrow shows possible commonly conserved genetic fragment (ccgf). (c) Partial sequences of ccgfs. Point mutations are shown with red letters. Completely matching regions are shown with lines. (d) Clustering tree for nine samples. Here, tentative microbial species are assigned from the sequence obtained for ccgf. The tree was constructed using Phylip 3.69 and MEGA 5.1 software.