Sufficient amounts of DNA were available for exome-sequencing from 50 JMML patients (discovery cohort and validation cohort). Sequencing libraries were prepared at the DKFZ Genomics and Proteomics Core Facility using the Agilent “SureSelectXT Human all Exon V4” kit and subsequently sequenced on a HiSeq2000 instrument using the 100 bp paired-end mode. Alignment to the hs37d5 reference genome was performed using BWA-MEM63 (link). SAMtools/BCFtools (version 0.1.19) were used for single nucleotide variants (SNV) calling64 (link). SNV found with high frequency in other mutation databases (i.e., ‘common = 1’ tag in dbSNP or > 1% frequency in ExAC 0.3.1) were filtered out and only high-confidence mutations in the coding regions were kept. Calling of small insertions/deletions (INDEL) was performed by Platypus 0.8.1 using the same filtering process as with the SNV calling65 (link). Both SNV and INDEL calling were performed without using paired germline control samples. The data were plotted as an OncoPrint using the ComplexHeatmap package66 (link). Statistics between groups were performed using the non-parametric Wilcoxon's test.
Lipka D.B., Witte T., Toth R., Yang J., Wiesenfarth M., Nöllke P., Fischer A., Brocks D., Gu Z., Park J., Strahm B., Wlodarski M., Yoshimi A., Claus R., Lübbert M., Busch H., Boerries M., Hartmann M., Schönung M., Kilik U., Langstein J., Wierzbinska J.A., Pabst C., Garg S., Catalá A., De Moerloose B., Dworzak M., Hasle H., Locatelli F., Masetti R., Schmugge M., Smith O., Stary J., Ussowicz M., van den Heuvel-Eibrink M.M., Assenov Y., Schlesner M., Niemeyer C., Flotho C, & Plass C. (2017). RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia. Nature Communications, 8, 2126.
ExonGenome Germline Human Insertions deletions Jmml Mutations NucleotidePatients Platypus Same
Corresponding Organization : German Cancer Research Center
Other organizations :
University Medical Center Freiburg, University of Freiburg, University Hospital Heidelberg, Hospital Sant Joan de Déu Barcelona, Ghent University Hospital, St Anna Children's Hospital, Medical University of Vienna, Aarhus University Hospital, Bambino Gesù Children's Hospital, University of Pavia, University of Bologna, University Children's Hospital Zurich, Our Lady's Hospital, Children's Health Ireland at Crumlin, University Hospital in Motol, Charles University, Wroclaw Medical University, Princess Máxima Center
Exome-sequencing of DNA samples from 50 JMML patients
dependent variables
Single nucleotide variants (SNVs)
Small insertions/deletions (INDELs)
control variables
Hs37d5 reference genome used for alignment
SAMtools/BCFtools (version 0.1.19) used for SNV calling
Platypus 0.8.1 used for INDEL calling
Filtering of high-frequency SNVs found in other mutation databases
Filtering of both SNVs and INDELs without using paired germline control samples
Annotations
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