However, to our knowledge, this type of technique has not been applied to profiling complex microbial communities to date. Here, we tested a set of padlock probes to evaluate the potential of the method for AD process monitoring and more generally for microbial community analysis (Figure 4). In order to establish the functionality

and target sequence specificity of the probes, we used 10 fmol of probe-specific synthetic dsDNA oligos as templates for the probe pool in ligation reactions. Signals from the subset of probes corresponding to the templates present in each pool could be clearly distinguished from signals from the rest of the probes (Additional file 4), suggesting a good target sequence specificity. However, the signal intensities of different probes varied considerably at the constant 10 Quisinostat fmol template concentration, probably

because of random variability of PCR [72] and sequence bias of ligation [73, 74]. Approximately 10% of the probes were not functional despite their perfect alignment to template. Six probes were non-specific giving false positive signals, despite that they did not have good alignment to any of the templates. To estimate the amount of detectable template, we tested template pools each containing 24 templates, at four different concentrations each. The probe signal intensities correlated with concentration (Additional file 5) with the highest concentration (1 fmol/μl/template) giving the highest signals while at the lowest concentration (0.001 fmol/μl/template) practically

none of the probes produced detectable signals. Almost all of the probes had GS-1101 in vivo consistently lower signals with lower concentrations and the majority of probes were still detectable at 0.01 fmol/μl/template concentration, suggesting that the method may be used for semiquantitative assaying over at least three orders of magnitude. Figure 4 Comparison of sequencing, microarray and qPCR. Performance of probe A123 on Megestrol Acetate samples M1, M2, M3 and M4. (a) Relative abundance of sequencing reads corresponding to microarray probe A123 bacterial target groups, (b) microarray signal intensities and (c) TaqMan assay using the same probe sequence. Microarray analysis of the AD samples To evaluate the microarray’s capability in analysing the AD samples, we performed ligation reactions using about 200 ng of non-amplified sample DNA as template for the probe pool. The microarray signals from the mesophilic samples M1 and M2 and the thermophilic samples M3 and M4 grouped separately and along the gradients of physical and chemical parameters in a similar way as with sequencing data (Figure 5) in redundancy analysis [16]. This suggests that our microarray had the ability to monitor changes in the microbial community structure in response to conditions of the digestor, an important aspect of in-process monitoring of AD status.