Whole genome sequencing of KrasG12D-driven mouse primary pancreatic cancer cell culture for bioinformatic inference of chromothripsis

We frequently observed complex copy number patterns on chromosome 4, involving the Cdkn2a locus, in primary pancreatic cancer cell culture from a KrasG12D-driven mouse model. The regularity of oscillating copy number states in most cancers suggested chromothripsis as the predominant process underlying these complex alterations. To confirm this, we performed whole-genome sequencing of the primary pancreatic cancer cell culture S821. For estimation of copy number states, the Bioconductor HMMcopy package 1.16.0 was used followed by segmentation with the Bioconductor DNAcopy package 1.48.0. For LOH analysis variant positions in control and tumor were computed with samtools mpileup v1.3.1. Only positions in regions with mapping quality of 60 and an average phredscore of 20 were considered for further analysis. Furthermore, positions harboring strand bias and variant allele frequencies less than 20% and above 85% in the control were excluded as they are likely homozygous in the germline. The minimal cutoff coverage for a given polymorphic position in the control was set to eight reads. Segmental duplications (UCSC Genome Browser) and regions with mouse line specific variation (Mouse Genomes Project, REL-1505) were excluded. For this set of SNPs the difference of frequencies between tumor and control samples were calculated. DELLY v0.7.6 was used for calling structural variations (SVs). SV-classes were defined according to DELLY callings: Deletion-type (3to5), duplication-type (5to3) and inversion-type (5to5 and 3to3). The predicted rearrangements were merged and filtered based on variant frequency, mapping quality and the distance between two connected breakpoints. The existence of chromothripsis was tested by applying the six hallmark criteria proposed by Korbel et al. (Cell, 2013). Clustering of SV breakpoints was tested using a ?²-goodness-of-fit test. Regularity of oscillating copy number states in the chromothriptic model was compared to a virtual chromosome generated by a Monte Carlo simulation, as described in Stephens et al. (Cell, 2011). For each distinct number of breakpoints, 100 simulation runs were completed and mean values as well as 95% CI were calculated. Interspersed loss and retention of heterozygosity was analyzed by calculating the Jaccard index between heterozygously deleted segments and regions comprising LOH and SNP information. Randomness of observed DNA segment order was tested using a Monte Carlo simulation as described in Korbel et al. (Cell, 2013).
The uniform distribution of SV-types was tested using a ?²-goodness-of-fit test.
The Wald-Wolfowitz runs test as implemented in R package randtests 1.0 was performed for testing right-sided against the null hypothesis of randomly distributed 5'-to-3' breakpoint joints sequence.