The first part of the sample handling process is extracting the dna from the sample. This involves a process known as Repeated Bead Beating. 

This process destroys the outer cell walls of the bacteria and enables us to extract the DNA into a buffer solution. This solution containing islolated DNA is measured for DNA concentration and sample quality. These sample characteristics are then logged.

Microbial marker sequences are amplified using polymerase chain reaction (PCR) protocols and sequenced with the Illumina MiSeq platform.

These are standard techniques used in molecular biology to amplify a segments of DNA across several orders of magnitude. For bacterial profiling the 16S rRNA gene is targeted while for fungal profiling the intergenic transcribed spacers (ITS) are used. The 16S rRNA gene and ITS sequences are universal marker sequences, which act as molecular clocks, and are used to relate the microbes in the samples to each other and to known reference strains.

For bacterial profiling a single step PCR protocol targeting the V3-V4 region of the 16S rRNA gene is employed (Ref: Kozich 2013). For fungal profiling a twostep PCR protocol targeting the Intergenic transcribed spacer (ITS1) is used.

While it has been common practice to generate operational taxonomic units (OTU) by clustering the obtained sequences at some set dissimilarity criteria, recent advances in data processing have given us the opportunity to infer the true amplicon sequence variants (ASV).

This state-of-the-art bioinformatic data analysis allows for discrimination of variants at single nucleotide level, allowing for the best phylogenetic resolution for further microbiota analysis.

This processing is used by Microbiota Center Amsterdam. It is a fast and accurate sample inference from amplicon data with single-nucleotide resolution.