16S fusion primers used in this study
Figure S1. 16S Fusion primers developed in this study. They include flow cell and sequencing primer binding regions for current Illumina sequencers. The amplified fragment has a size fo ~157 bp and can be sequenced directly after purification (one step PCR). Up to 10 samples can be uniquely tagged from forward and reverse direction and pooled in one NextSeq run. The bases used for shifting on Ins_F and Ins_R can be used to uniquely tag samples (inline barcodes). It is recommended that all 10 primer pairs are used in the following combination to maximize sequence diversity and reduce effects of tag switching by uniquely tagging samples from both sides: P5_Ins_R0+P7_Ins_F4, P5_Ins_R1+P7_Ins_F3, P5_Ins_R2+P7_Ins_F2, P5_Ins_R3+P7_Ins_F1, P5_Ins_R4+P7_Ins_F0, P5_Ins_F0+P7_Ins_R4, P5_Ins_F1+P7_Ins_R3, P5_Ins_F2+P7_Ins_R2, P5_Ins_F3+P7_Ins_R1, P5_Ins_F4+P7_Ins_R0
Distribution of reads obtained by NextSeq and number of reads discarded throughout the different bioinformatics processing steps
Figure S2. Number of sequences obtained per sample after library demultiplexing (A) and percentage of sequences excluded in different bioinformatic analysis steps (B). A: Library demultiplexing; Numbers above bars indicate the relative contribution (in percent) to the total number of sequences obtained for each sample. Sequencing started with Ins_F (white) or Ins_R (black) is indicated by bar color. B: Number of reads excluded in data processing steps. Mean percentage of sequence abundance in each processing step is written in brackets. Ins_F / Ins_R primer bias was tested with a t-test.