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Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer - PubMed

  • ️Sun Jan 01 2012

. 2012 Feb 17;148(4):780-91.

doi: 10.1016/j.cell.2011.11.065.

Ole B Schulz-Trieglaff, Zemin Ning, Ludmil B Alexandrov, Markus J Bauer, Beiyuan Fu, Matthew Hims, Zhihao Ding, Sergii Ivakhno, Caitlin Stewart, Bee Ling Ng, Wendy Wong, Bronwen Aken, Simon White, Amber Alsop, Jennifer Becq, Graham R Bignell, R Keira Cheetham, William Cheng, Thomas R Connor, Anthony J Cox, Zhi-Ping Feng, Yong Gu, Russell J Grocock, Simon R Harris, Irina Khrebtukova, Zoya Kingsbury, Mark Kowarsky, Alexandre Kreiss, Shujun Luo, John Marshall, David J McBride, Lisa Murray, Anne-Maree Pearse, Keiran Raine, Isabelle Rasolonjatovo, Richard Shaw, Philip Tedder, Carolyn Tregidgo, Albert J Vilella, David C Wedge, Gregory M Woods, Niall Gormley, Sean Humphray, Gary Schroth, Geoffrey Smith, Kevin Hall, Stephen M J Searle, Nigel P Carter, Anthony T Papenfuss, P Andrew Futreal, Peter J Campbell, Fengtang Yang, David R Bentley, Dirk J Evers, Michael R Stratton

Affiliations

Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer

Elizabeth P Murchison et al. Cell. 2012.

Abstract

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

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Graphical abstract
Figure 1
Figure 1

Tasmanian Devil Facial Tumor Disease Tasmanian devil facial tumor disease (DFTD) is a single cancer lineage spread by the horizontal transfer of living cancer cells.

Figure 2
Figure 2

Variation in Tasmanian Devil Normal and Cancer Genomes (A) Location, year of isolation, and karyotypes for 87T and 53T DFTD cancer cell lines. (B) Four genomes were sequenced in this study, two normal Tasmanian devil genomes (female and male) and two DFTD cancer genomes (87T and 53T). DFTD originated in the DFTD founder devil, and 87T and 53T are both clonally derived from their most recent common ancestor tumor (the progenitor tumor). The female normal sequence was used to assemble the Tasmanian devil reference genome. The number of substitutions and indels compared with the reference sequence is indicated for each genome. The number of variants that were unique to each genome is indicated in brackets. The number of variants in the most recent common ancestor tumor was inferred using the variants that were common between 87T and 53T. (C) Forward chromosome painting for the normal female fibroblast cell line carrying trisomy 6 that was used to generate the reference genome assembly. ∗ indicates a region of overlap between chromosomes 1 and 2 that was present in the metaphase image that was used to generate the karyotype. Cytogenetic comparison between Tasmanian devil and opossum is summarized in Figure S2. (D) Forward chromosome painting for the 87T DFTD tumor. (E) Reverse painting was performed by flow sorting 87T chromosomes to produce paints (labeled A to G and I to M) and hybridizing these with normal Tasmanian devil metaphases. The F paint includes two similarly sized 87T chromosomes that we were unable to separate with flow cytometry. (F) Summary of copy number variation in 87T DFTD genome (including only changes >10 Mb in size). See Figure S3 for complete 87T and 53T copy number data. See also Figures S2 and S3.

Figure 3
Figure 3

DFTD Origin (A) Y chromosome gene SRY is not detectable in DFTD using PCR. Primer sequences are available (Lachish et al., 2011; Murchison et al., 2010). (B) Number of X chromosome variants in female and male normal devil genomes and 87T and 53T DFTD genomes. Variants from a poorly assembled region at the end of chromosome X were excluded from this analysis. (C) Phylogenetic tree of devil mitochondrial variation. Each dot on the map indicates an individual devil and the color of the dot represents the mitochondrial haplotype for each devil. Each haplotype is also represented on the phylogenetic tree. DFTD mitochondrial haplotypes are indicated in gray; some DFTD tumors also had the haplotype represented by the red dot. See also Figure S3 for chromosome X copy number plots for 87T and 53T.

Figure 4
Figure 4

Somatic Evolution of DFTD (A) Nonsynonymous to synonymous ratios for variants occurring in genes in DFTD and in normal devil genomes and for variants inferred in the most recent common ancestor tumor of 87T and 53T (the progenitor). Only variants that were unique to the respective genomes were included in the analysis. Nonsynonymous gene variants in DFTD are listed in Table S4. (B) Heterozygosity for variants unique to the normal male genome, DFTD genomes and inferred in the most recent common ancestor tumor of 87T and 53T (the progenitor). (C) Mutation spectrum of single-base substitutions in DFTD and normal devil genomes. Only variants that were unique to the specified sample(s) were included in the analysis. The spectrum and ratios of the most recent common ancestor (progenitor) tumor (which includes the germline variants of the founder devil) were calculated using the common variants between 87T and 53T that were not present in the normal devil genomes. (D) Copy number analysis of Tasmanian devil chromosome 3 in 53T and 87T. Each dot represents the log2 ratio (that falls within the range −2 to +2) between the number of sequence reads in the tumor genome and the number of sequence reads in the female normal genome that align within a 2 kb genomic window. If p < 1 × 10−5, the dot is red; otherwise, dots are gray. Homozygous variants unique to either 53T or 87T are shown as black dots above the copy number plot. See Figure S3 for genome-wide comparison of 87T and 53T copy number. (E) Structural variants unique to 87T and 53T. Each chromosome is represented by a colored bar and black lines indicate either large-scale rearrangements (connecting lines) or small-scale rearrangements (single lines). Three 87T rearrangements that occurred close together on chromosome 2 are represented with a single bar (∗). See Table S3 for rearrangement coordinates. See also Figure S3 and Tables S3 and S4.

Figure 5
Figure 5

DFTD Clonal Dynamics Phylogenetic tree summarizing genetic variation found in 104 DFTD tumors collected from 69 Tasmanian devils. The tree was constructed using both nuclear and mitochondrial variants and branch length represents the number of variants (either nuclear or mitochondrial) that distinguish each tumor type from the most likely ancestral tumor type (solid gray). Trapping locations for devils captured with DFTD are indicated either on the map of Tasmania (top) or on the map of the Forestier Peninsula (bottom), with colors indicating the genetic subgroup to which each animal's tumor(s) belongs. Four Forestier Peninsula tumors for which trapping location data were not available are indicated in boxes. The six cases in which a single devil had multiple tumors with more than one genotype are represented on the map with just one genotype. See also Figure S4 for further details about devils with multiple tumors and Table S5 for genome coordinates for variants.

Figure S1
Figure S1

Comparative Genome Size in Tasmanian Devil, Human and Chicken, Related to Table 1 Univariate plot of a mixed nuclear suspension containing chicken erythrocyte nuclei, Tasmanian devil leukocyte nuclei and human leukocyte nuclei stained with propidium iodide. The DNA content is displayed as a histogram. DNA analysis revealed three separate peaks on the plot; C (chicken erythrocyte nuclei), TD (Tasmanian devil) and H (human). The ratio of Tasmanian devil to human DNA content is displayed inset on the plot.

Figure S2
Figure S2

Cross-Species Cytogenetic Comparison between Devil and Opossum, Related to Figure 2 (A) Chromosome correspondence between Tasmanian devil and opossum established by hybridizing Tasmanian devil chromosome-specific paint probes onto opossum metaphases. Lines connect each devil chromosome with the opossum chromosome with which it predominantly hybridized. Devil chromosomes 2 and 3 were each homologous to two opossum chromosomes, illustrated with blue and red lines respectively. (B) Flow cytometry analysis of Tasmanian devil and opossum chromosomes. The red line marks the division between Tasmanian devil chromosomes (above the line) and opossum chromosomes (below the line). This shift toward the Hoechst axis in the Tasmanian devil chromosomes relative to opossum indicates greater A+T content in Tasmanian devil. Peaks for chromosomes with one to one correspondence between devil and opossum are connected with red arrows.

Figure S3
Figure S3

Genomic Copy Number Variation in 87T and 53T, Related to Figures 2, 3, and 4 Separate plots are shown for each chromosome (chromosomes 1 to X). Each dot represents the log2 ratio between the number of sequence reads in the tumor genome and the number of sequence reads in the female normal genome that align within a 2 kb genomic window. If p < 1 × 10-5, dot is red, otherwise dots are gray. Windows which contained no reads in the tumor (i.e., putative homozygous deletions) are represented with a red dot at log2 ratio of −4.

Figure S4
Figure S4

Analysis of Multiple DFTD Tumors from the Same Host, Related to Figure 5 Six Tasmanian devils (Devil IDs 40, 51, 53, 66, 70 and 101) were identified with two more or more DFTD tumors with different genotypes. In three of these cases (Devils 40, 53, 66), one of the tumors had a new genotype that had not been identified in any other animal (arrows on phylogenetic tree). In the remaining three cases (Devils 51, 70 and 101) the second genotype could be identified in other animals. The locations where the six devils with non-matching genotypes were trapped are indicated on the map. All DFTD cases were confirmed by genotyping, and any tumor with unacceptably high levels of contaminating host DNA was excluded from the analysis. The corresponding genotypes for each devil are listed in Table S5.

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