Miseq sequencing device

Genomic DNA of C. glutamicum cells was purified using the NucleoSpin® Microbial DNA Kit (Macherey Nagel). About ~100 mg cell pellet (from ~1 ml BHI-overnight culture) yielded approx. 10 µg DNA. In total, 4 µg of genomic DNA was used for library preparation and indexing with the TruSeq DNA PCR-free sample preparation kit (Illumina). Quantifications of the resulting libraries were conducted using KAPA library quant kits (Peqlab) and were normalized for pooling. A MiSeq sequencing device (Illumina) was used for paired-end sequencing with a read-length of 2 × 150 bases. Data analysis and base calling were accomplished with the Illumina instrument software and stored as fastq output files. Obtained sequencing data were imported into CLC Genomics Workbench (Qiagen Aarhus A/S) for trimming and base quality filtering. The output was mapped to accession BX927147 as the C. glutamicum ATCC 13032 reference genome^{34 (link)} or CP005959 as the reference genome for MB001^{19 (link)}. The resulting mappings were used for the quality-based SNP/variant detection with CLC Genomics Workbench. The detected SNPs were manually inspected regarding their relevance.

The index patient III‐1 and her parents (Figure 1a) were counseled at the Center for Hereditary Breast and Ovarian Cancer, University Hospital of Cologne, Germany. Physicians qualified in genetic counseling recorded personal and family cancer history, information regarding age at first diagnosis, tumor receptor status, and treatment of the index patient. Written informed consent was obtained from all individuals, and ethical approval was granted by the ethics committee of the University of Cologne (07‐048). Genomic DNA was isolated from venous blood samples. Next generation sequencing technology was applied using an Illumina MiSeq sequencing device (Illumina, San Diego, USA) and the customized diagnostic TruRisk® multigene panel for target enrichment (Agilent, Santa Clara, USA) covering BRCA1, BRCA2, and additional breast/ovarian cancer predisposition genes (ATM, BRIP1, CDH1, CHEK2, PALB2, RAD51C, RAD51D, TP53). Variant classification was performed in accordance with the regulations of the international ENIGMA consortium (Evidence‐based Network for the Interpretation of Germline Mutant Alleles; https://enigmaconsortium.org; version 1.1:26 March 2015).

HLA typing was performed using commercial test kits, the Alltype NGS 11-Loci (One Lambda, West Hills, CA, USA) and AlloSeq^® Tx 17 (CareDx, San Francisco, CA, USA). The sequencing was performed on a MiSeq Sequencing device (Illumina, San Diego, CA, USA), strictly following the manufacturer’s recommendations. The laboratory in Leipzig that performed the HLA NGS typing has a European accreditation for NGS HLA typing.