E01-068-01 Explanation standard 16S-ITS microbial profiling analysis V3 Taxonomic classification pipeline OVERVIEW OF ANALYSIS STEPS INVOLVED IN THE TAXONOMIC CLASSIFICATION PIPELINE Short paired sequence reads were generated using the Illumina RDP 16S rRNA gene databases. Based on the alignment scores of the MiSeq system and converted into FASTQ files using the BCL2FASTQ pseudoreads, the taxonomic classes were assigned by associating pipeline version 2.18. The Illumina paired reads were merged into each pseudoread to the best matching taxon. The taxonomic depth single reads (so-called pseudoreads) through sequence overlap, after of the lineage is based on the identity threshold of the rank; Species removal of the forward and reverse primers. Chimeric pseudoreads 99%, Genus 97%, Family 95%, Order 90%, Class 85%, Phylum 80%. were removed and the remaining reads were aligned to the A brief overview of the pipeline is given in the figure below. Q-check Target amplification Illumina MiSeq Pseudoreads through (e.g. 16S rRNA, ITS) sequencing sequence overlap Interactive visualization Taxonomic classification Chimera removal in online portal based on DNA databases MORE INFORMATION 1 Ribosomal Database Project: data and tools for high throughput rRNA analysis. Cole JR, Wang JA et al. (Nucl Acids Res. 2014). PO Box 1336 T +31 (0)71 523 39 17 2302 BH Leiden E
[email protected] The Netherlands W baseclear.com Metagenomics online analysis portal OVERVIEW PER PROJECT: TREE AND BAR-CHART In the project overview, a tree is been constructed on-the-fly: samples that share a similar taxonomic composition displayed in close proxi- FIGURE INTERPRETATION mity.