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. 2023 Mar 22;3(4):100281.
doi: 10.1016/j.xgen.2023.100281. eCollection 2023 Apr 12.

Long-read sequencing of diagnosis and post-therapy medulloblastoma reveals complex rearrangement patterns and epigenetic signatures

Tobias Rausch  1   2 , Rene Snajder  3   4   5 , Adrien Leger  6 , Milena Simovic  7 , Mădălina Giurgiu  8   9 , Laura Villacorta  2 , Anton G Henssen  10   8   11 , Stefan Fröhling  12   13   14 , Oliver Stegle  1   3   15 , Ewan Birney  6 , Marc Jan Bonder  3 , Aurelie Ernst  7 , Jan O Korbel  1   6   16
Affiliations

Long-read sequencing of diagnosis and post-therapy medulloblastoma reveals complex rearrangement patterns and epigenetic signatures

Tobias Rausch et al. Cell Genom. .

Abstract

Cancer genomes harbor a broad spectrum of structural variants (SVs) driving tumorigenesis, a relevant subset of which escape discovery using short-read sequencing. We employed Oxford Nanopore Technologies (ONT) long-read sequencing in a paired diagnostic and post-therapy medulloblastoma to unravel the haplotype-resolved somatic genetic and epigenetic landscape. We assembled complex rearrangements, including a 1.55-Mbp chromothripsis event, and we uncover a complex SV pattern termed templated insertion (TI) thread, characterized by short (mostly <1 kb) insertions showing prevalent self-concatenation into highly amplified structures of up to 50 kbp in size. TI threads occur in 3% of cancers, with a prevalence up to 74% in liposarcoma, and frequent colocalization with chromothripsis. We also perform long-read-based methylome profiling and discover allele-specific methylation (ASM) effects, complex rearrangements exhibiting differential methylation, and differential promoter methylation in cancer-driver genes. Our study shows the advantage of long-read sequencing in the discovery and characterization of complex somatic rearrangements.

Keywords: Nanopore methylation calling; cancer genomics; chromothripsis; complex rearrangements; epigenetic signatures; long read sequencing; templated insertions.

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Conflict of interest statement

E.B. is a paid consultant and shareholder of ONT. A.L. has received financial support from ONT for consumables during the course of the project and is currently an employee of ONT. O.S. is a paid consultant of Insitro, Inc.

Figures

None
Graphical abstract
Figure 1
Figure 1
Haplotype-phased assembly of an inter-chromosomal chromothripsis event (A) A circos plot of the primary tumor showing, from outside to inside, the chromosome ideograms, read-depth, large (>10 Mbp) SVs, and inter-chromosomal rearrangements: orange, deletion-type; violet, duplication-type; light green, head-to-head inversion-type; pink, tail-to-tail inversion-type; dark green, inter-chromosomal. (B) Chromosome 5 exhibits a pattern of oscillating copy-number states (lower panel) and alternating heterozygous allele frequencies (upper panel) common to chromothripsis. (C) The CS11-17 assembly contains two contigs with amplified segments from chromosome 11 and chromosome 17. Segments from chromosome 11 are in green, segments from chromosome 17 in purple. The part of the chromosomes displayed (1–50 Mbp) is shown with a gray background in the chromosome ideograms as well as the locations of the amplified segments (green and purple segments). (D) A chained alignment of contig 1 of the CS11-17 assembly against GRCh38. Forward matches are in blue, reverse matches in ochre. Matches are clustered within 1 Mbp, and distinct alignment regions are separated by a vertical gray line. Numbers 1–5 correspond to amplicons labeled as 1–5 in (C). (E) FISH analysis identifies potential marker or ring chromosomes associated with the CS11-17 structure by means of localized signals of the red RP11-651L9 probe (chr17:16,169,409–16,359,715), shown in the left two panels, and the green centromere 17 probe, shown in the right two panels. The boxed structure (yellow) contains a putative ring or marker chromosome with enlarged views in the outer panels.
Figure 2
Figure 2
TI threads (A) Self-alignment of a single ONT read that spans the entire length of the TI thread, displaying an array of repetitive short sequence matches reflecting the copying and concatenation of few source sequence segments. (B) Matched Illumina data show a characteristic coverage increase (upper panel) in Integrative Genomics Viewer (IGV). An alignment of the ONT read (y axis) against selected TI source sequences (x axis) shows how the ONT read aligns across these source sequences multiple times in seemingly random order. (C) A scheme showing how TIs are copied and pasted in direct adjacency and random order into a growing TI thread. Arrows next to the TI thread indicate the segment orientation and dashed lines show discovered adjacencies among individual TIs. (D) The colocalization of the beginning and the end of the TI thread (purple arrow) with chromothripsis segments on chromosome 5 (left) and chromosome 7 (right). (E) Analysis of 2,569 cancer genomes reveals that liposarcomas often harbor TI threads, preferentially on chromosome 12 (main panel). The inset shows the distribution of TIs along chromosome 12 where each horizontal line is a distinct liposarcoma sample. (F) A liposarcoma validation sample (P1) sequenced using long reads confirms the TI thread signature. Chained alignment matches to GRCh38 are shown for a single ONT read with forward matches in blue and reverse matches in ochre. Aligned segments show strong coverage increases in the matched Illumina short-read data (top panel; IGV28) with SV-supporting reads and soft-clips.
Figure 3
Figure 3
Functional analysis of primary tumor and relapse sample (A) Number of CpGs in regions found to be differentially methylated in the sample comparison (primary tumor vs relapse) as well as ASM in the two samples. Colors represent an estimation of discoverability with short-read sequencing methods. CpGs in low-complexity regions (soft-masked in reference) are more difficult to map using only short reads. CpGs not phaseable with short reads are further than 150 bp from a phased heterozygous non C>T variants. (B) Methylation of NRN1 promoter and enhancer in the primary tumor sample. (C) Heterozygous deletion in promoter of PTCH1 (tumor-suppressor gene and driver in medulloblastoma) with differential methylation in the remaining haplotype. (D) Predicted gene fusion pairs from Arriba validated using ONT long-read information, thresholded by confidence as reported by Arriba. Fusion pairs in the “supported by individual reads” category are supported by at least one genomic read with a chimeric alignment including both genes. Pairs in the “explainable using genomic breakpoints” category have a plausible explanation by following a graph of structural variations that connect the two genes. The category “high confidence read support” refers to pairs where both these criteria are met. (E) Example of a gene fusion pair that can be explained using genomic breakpoints but with no individual genomic read that covers both genes. Two separate insertions of a total length of 42,797 bp appear to be involved in the fusion of LINC01091 and FKBP9 such that, even in ONT reads, there was no read extending across the entire gene fusion. (F) PCDH17 (tumor-suppressor gene) promoter with ASM pattern in the primary tumor sample.
Figure 4
Figure 4
Methylation of complex genomic rearrangements (A) Methylation rates of chromothriptic contig CS11-17 in the primary tumor sample show global demethylation of contig 2, containing genes TRIM66 and STK33, to a methylation rate of 42% on the CS11-17 haplotype from 76% in the corresponding genomic ranges on the non-chromothriptic haplotype. While contig 1 displays some allele-specific differences, no significant global effects are detected. (B) ASE correlates with promoter-linked ASM in primary tumor (Pearson R, −0.38, p value 6.6 × 10−3 for all ASM effects displayed and nominally significant ASE effects, Pearson R, −0.61, p = 5.1 × 10−3 when considering only ASM with >0.5 absolute methylation rate difference). (C) Demethylation of CS11-17 haplotype of contig 2 effect shown on TRIM66 promoter. (D) ASM of promoter of gene PLEKHM1 on contig 1.
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