title: microbiome of ixodes ricinus: adult male
design: Illumina 16S rRNA gene amplicon libraries (Illumina, SanDiego, CA, USA) were generated and sequenced at BaseClear(Leiden, The Netherlands). In short, barcoded ampliconsfrom the V3-V4 region of 16S rRNA genes were generatedusing a 2-step approach. Ten to 25 ng genomic DNA wereused as a template for the first PCR with a total volume of50ml using the 341F (59-CCTACGGGNGGCWGCAG-39)andthe 785R (59-GACTACHVGGGTATCTAATCC-39) primersappended with Illumina adaptor sequences. PCR productswere purified and the size of the PCR products was checkedon a Fragment Analyzer (Agilent Technologies, Santa Clara,CA, USA) and quantified by fluorometric analysis. PurifiedPCR products were used for the second PCR in combinationwith sample-specific barcoded primers (Nextera XT IndexKit; Illumina). Subsequently, PCR products were purified andchecked on a Fragment Analyzer. After quantification, mul-tiplexing was performed, followed by sequencing on anIllumina MiSeq System with the paired-end (23) 300-bpprotocol and indexing. The sequencing run was analyzedwith the Illumina Casava pipeline (v.1.8.3;http://cancan.cshl.edu/labmembers/gordon/fastq_illumina_filter/) with demultiplexingbased on sample-specific barcodes. The produced raw sequenc-ing data was processed by removing the sequence reads of toolow quality (onlypassing filterreads were selected) anddiscarding reads containing adaptor sequences or PhiX con-trol with an in-house filtering protocol. A quality assessmenton the remaining reads was performed using the FastQC qualitycontrol tool v.0.10.0 (Bioinformatics;https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).