Fig. 2 Further analysis of inter-and intra-chromosomal rearrangements in the two cell lines a) Circos plot of A549 b) KRAS SNPs c) KRAS duplication in H441 d) Circos plot of H441
To detect SVs we processed the alignments with the software tool sniffles, discarding any SVs that were not supported by five or more reads. We created Circos plots for each cell line (Fig 2). A large number of inter- and intra-chromosomal rearrangements can be seen. Additionally, both cell lines are known to have non-synonymous mutations in the KRAS gene on chromosome 12, in adjacent positions. A549 carries a C -> T transition in position 25245351 (G12S) and H441 carries a C -> A transversion in position 25245350 (hg38 coordinates). These mutations are clearly visible in the sequence data (Fig. 2b). The C -> A mutation in H441 was detected at a ratio of approximately 2:1 in our reads. Consistent with this, chromosome 12 appears to be triploid in the published H441 karyogram. Fig 2d shows normalised coverage in 1 kb bins for both cell lines for the 12p12.1 region of chromosome 12. The KRAS gene and its surrounding area has higher normalised coverage in cell line H441 compared to A549, supporting this interpretation. The full extent of the duplication can be seen clearly, and a further, smaller, duplication is visible within that duplication. These results show the power of long nanopore sequence reads for the analysis of tumour genomic DNA, and the identification of SVs with far higher resolution than karyotyping.