Basespace online analysis tool

A multiplex-PCR approach (Truseq Amplicon Cancer Panel, Illumina) of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes was used to identify mutations in a cohort of patients with MPN (S4 Table). The manifest file with distinct data of all primers/amplicons can be downloaded at http://support.illumina.com/downloads/truseq_amplicon_-_cancer_panel_manifest_file.html. 5μl (250ng) of gDNA of each sample was prepared according to the TruSeq sample preparation guide. Data were analyzed using MiSeq reporter (Illumina), BaseSpace online analysis tool (Illumina) and SeqPilot software. To avoid scoring of mutants that were detected as a result of unfaithful PCR amplification or sequencing, a sequence variant was only further analyzed when the following conditions were met: (1) the absolute coverage at the variant site was ≥50 (2) the variant was detected in at least 10 (absolute) reads, and (3) the variant was detected in 5% of all reads at the variant site. For potential pathogenic effects of new detected variants, additional analysis was performed using MutationTaster software (http://www.mutationtaster.org) and PolyPhen-2 software (http://genetics.bwh.harvard.edu/pph2/bgi.shtml).

The genotype of four patients has already been published before [27 (link)-29 (link)]. For the other patients we utilized a multiplex-PCR approach (Truseq Amplicon, Illumina^®) if enough DNA was available. To this end, we analyzed 158 amplicons covering 8 telomerase associated genes (NOP10, NHP2, CTC1, DKC1, TERT, TERC, TIN2, RTEL1). 250ng of genomic DNA of 9 DKC samples was prepared according to the TruSeq sample preparation guide. Sequencing data were analyzed using BaseSpace online analysis tool (Illumina, San Diego, CA, USA) and SeqPilot software (JSI medical systems, Ettenheim, Germany). To detect heterozygous or homozygous variants, conditions for scoring a mutant allele were: absolute coverage at SNV-Site ≥ 50 and relative allele burden of the variant ≥ 30% of all reads. Further analysis was performed using MutationTaster software (http://www.mutationtaster.org) and PolyPhen-2 software (http://genetics.bwh.harvard.edu/pph2/bgi.shtml) to predict pathogenicity of detected variants.