## 16S rDNA amplicon sequencing 

The bacterial population in the TIM-2 samples were analyzed using Next Generation sequencing. DNA from TIM-2 lumen samples was isolated and purified for universal 16S rDNA amplification targeting the V4 region with universal primers. 

Subsequently these amplicons were further processed for amplicon sequencing on the MiSeq system. The sequencing results from the MiSeq system were processed in a dedicated data processing pipeline. To this purpose, the Mothur pipeline is implemented in combination with the Silva database which provides comprehensive, quality checked and regularly updated databases of aligned small 16S rRNA sequences (https://www.arb-silva.de/). 

In brief, processing of the sequencing data using the Mothur pipeline is a computer- based script that comprises in summary: quality checks, normalization and identification of the sequences at taxon level 6 (genus level) by using the Silva data base (a database with 16S rRNA sequences linked to taxonomic information). Based on frequencies of the presence of specific sequences, the bacterial community composition can be described. This yields a community profile which is presented in a bar plot indicating the relative abundance of the various bacterial genera present. The bar plots give an overview on all bacterial genera present and identified, with exception of nonclassified bacterial genera, which are indicated as unknown genus but with a lower level identification (taxon or family for instance). 

## Data analysis 

The metabolite concentration as measured in the different samples (lumen and dialysate over time) was multiplied by the volume of the respective sample. For the dialysates, the total volume of each sample was measured. For the lumen samples, a theoretical volume (same for all TIM-2 units and at every sampling time) was used to calculate the absolute amount of metabolites. 

The SCFA, BCFA, and ammonia values from t=0h were used to set the values of these metabolites in the TIM-2 system artificially to zero. 

The graphs in the report depict the total (cumulative) produced amount (luminal and dialysate samples) over time. 

