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Targeted resequencing of single cell-derived colonies

Individual single cell-derived clones DNA will undergo multiplexed barcoded capture

and sequencing (>500x) on the custom capture set in order to provide a low-cost,

high-throughput assessment of the genetic diversity found within the individual

colonies. This will again be performed at the ICR TPU with bioinformatics support

within the Jones lab. Preliminary experiments have established the feasibility of this

approach and provided evidence of genetic diversity in colonies derived from single

cells of the DIPG sample. Private events including mutations in functionally relevant

genes such as those encoding receptor tyrosine kinases, histone methyltransferases

and DNA repair enzymes were discovered (Figure 3).

Figure 3 - Modelling subclonal variation of DIPG in vitro (a) Circos plot representing whole exome

sequencing of the tumour DIPG101. Somatic mutations are labelled on the outer ring, with copy

number changes (red = gain, blue = loss) on the inner rings. (b) Targeted resequencing of 435 genes

recurrently mutated in pGBM/DIPG applied to DIPG7 tumour and primary cell culture, as well as

multiple single cell-derived subclones. Variant allele frequency is plotted on the y axis. (c) Plot of variant

allele frequency versus log read depth for targeted resequencing of DIPG7 tumour and initial primary

culture. Gene mutations significantly different between the two are labelled - PIK3CA C420R present in

the tumour specimen is absent from the cells, which instead harbour H1047R. Heterozygous SFN

mutation from the tumour is homozygous in the cells. (d) Plot of variant allele frequency versus log read

depth for targeted resequencing of DIPG7 subclones. Subclone F10 harbours a heterozygous

SUV420H1 mutation, present as only 2/678 reads in the bulk cells from the original tumour specimen

and 0/64 in the high depth targeted resequencing of the primary DIPG7 tumour. For the tumour

samples and most subclones, H3F3A K27M is present at an allele frequency of 0.66 due to an extra

copy of the mutant allele as a result of chromosome 1q gain. In subclone D4 the wild-type allele is lost,

giving rise to a homozygous locus.

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