A Deterministic Analysis of Genome Integrity during Neoplastic Growth in <i>Drosophila</i>

<div><p>The development of cancer has been associated with the gradual acquisition of genetic alterations leading to a progressive increase in malignancy. In various cancer types this process is enabled and accelerated by genome instability. While genome sequencing-based analysis of tumor genomes becomes increasingly a standard procedure in human cancer research, the potential necessity of genome instability for tumorigenesis in <i>Drosophila melanogaster</i> has, to our knowledge, never been determined at DNA sequence level. Therefore, we induced formation of tumors by depletion of the <i>Drosophila</i> tumor suppressor Polyhomeotic and subjected them to genome sequencing. To achieve a highly resolved delineation of the genome structure we developed the Deterministic Structural Variation Detection (DSVD) algorithm, which identifies structural variations (SVs) with high accuracy and at single base resolution. The employment of long overlapping paired-end reads enables DSVD to perform a deterministic, i.e. fragment size distribution independent, identification of a large size spectrum of SVs. Application of DSVD and other algorithms to our sequencing data reveals substantial genetic variation with respect to the reference genome reflecting temporal separation of the reference and laboratory strains. The majority of SVs, constituted by small insertions/deletions, is potentially caused by erroneous replication or transposition of mobile elements. Nevertheless, the tumor did not depict a loss of genome integrity compared to the control. Altogether, our results demonstrate that genome stability is not affected inevitably during sustained tumor growth in <i>Drosophila</i> implying that tumorigenesis, in this model organism, can occur irrespective of genome instability and the accumulation of specific genetic alterations.</p></div

Coding sequences are less susceptible to SV accumulation.

<p>(A) Genome browser view depicting the concordant and discordant coverage of the control (blue) and the tumor (red) samples across two protein-coding genes, and identified SVs therein. The detected insertions and deletions localize outside of coding sequences, and affect introns, intergenic spaces and UTRs. (B) Genome-wide breakpoint distribution across distinct functional compartments. Different subsets of the genome were selected according to following characteristics: <i>genome</i> corresponds to the full-length genome; the <i>unique genes</i> do not share common positions with any other gene; <i>overlapping genes</i> are non-unique genes; <i>exonic</i> regions, containing <i>3′UTRs, 5′UTRs</i> and coding sequences (<i>CDS</i>) were obtained from the unique genes in order to avoid ambiguity; In addition, <i>intronic</i> and <i>intergenic</i> regions as well as donor/acceptor splice sites (<i>splice sites</i>) were considered. For each subset the number of contained breakpoints was computed and normalized to the total length.</p

Genomic context analysis can indicate mutational mechanisms causing SVs.

<p>(A) DNA sequence and insertion frequency of the 10 most commonly inserted sequences identified within the control genome. For the tumor the tenth most frequently inserted sequence corresponds to CA with 897 insertions. For the sake of a clear representation the eleventh most frequently inserted sequence (AAA, 894 insertions) is shown. (B) The fraction of single base insertions within simple repeats consisting of the same base type, computed with respect to all single base insertions. Simple repeats of a minimum length of 4 were considered. (C) A genome browser view of a genomic locus containing two insertions (I/V), two deletions (II/IV) and one tandem duplication (III). As indicated by the discordant coverage and horizontal bars, these high-confidence SVs are both identified within the tumor and the control genomes, and have therefore been inherited from the parental strains.</p

Summary of PCR-based validation experiments.

<p>SVs are considered to be confirmed whenever at least one aberrant allele was detected either within <i>ph</i>-RNAi induced tumors or the parental strains irrespective of the zygosity. The size range of a tandem duplication corresponds to a single duplication event. n =  Number of tested events; Het  =  expected to be heterozygous; Hom  =  expected to be homozygous; Small ins.  =  Small insertions; Tan. dupl.  =  tandem duplications.</p

Concordant and discordant read coverage reveal extensive similarity between the tumor and control.

<p>(A) A genome browser view of representative kb of the <i>Drosophila</i> reference genome. The tracks, denoted concordant and discordant, represent the total number of concordantly and discordantly aligning read pairs at a particular genomic position. (B) A browser view of homozygous and heterozygous SVs at higher resolution. In the case of homozygous SVs (I/II), the concordant coverage is decreasing to zero, as no wildtype allele is present anymore. In contrast, heterozygous events contain both a wildtype allele and an acquired SV, and are therefore characterized by a decrease within the concordant coverage to 50% (III). (C) Venn diagram representing the number of small insertions (of size bp) identified within the tumor and the control with weight (upper) and weight (lower), respectively. (D) Smoothed scatterplot representing the concordant coverage on the vertical axis and the discordant coverage (weight) on the horizontal axis for different subsets of small insertions. The three columns, from left to right, correspond to all small insertions (All), small insertions found in both genomes (Intersection) and small insertions specifically identified within the indicated genome (Specific), respectively.</p

Summary of the sequencing experiments and the seed-based alignment classification.

<p>Numbers refer to read pairs [million].</p

Depletion of <i>polyhomeotic (ph)</i> induces neoplastic tumors.

<p>A) Ph expression in the normal wing disc of third instar larvae (left) expressing the reporter <i>en-GAL4 </i> UAS-<i>myr-RFP</i>, UAS-<i>Dicer2</i>, <i>NRE:EGFP</i> (right). B) Downregulation of Ph induced by the RNAi reporter observed in the posterior compartment. Posterior compartment (red RFP) shows overproliferation phenotype (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087090#pone.0087090-Saj1" target="_blank">[26]</a>). C) Schematic outline of the experimental workflow. Eggs of the same genotype were developed at different temperatures. The tumor suppressor <i>ph</i> is specifically depleted (RNAi) at 25°C within the posterior compartment (p, RFP signal in red) of wing imaginal discs, leading to the formation of large tumors (upper). To allow for the accumulation of SVs, tumors are transplanted for a period of four weeks. At 18°C depletion of Ph is not sufficient to drive tumorigenesis (lower) and corresponding wing imaginal discs were used as control. Genomic DNA from both samples was isolated and subjected to paired-end sequencing. Notch-dependent EGFP expression (green) marks the boundary of the dorsal and ventral compartments. The white dashed outline marks the remnant anterior compartment (a) with normal Notch signaling along the dorsal/ventral boundary, while the grey dashed outline labels the haltere disc (hd).</p

Different directed edges belonging to . chr = chromosome.

<p>Different directed edges belonging to . chr  =  chromosome.</p

Schematic representation of the SV detection performed by DSVD.

<p>(A) Genomic rearrangements join different regions of the genome resulting in aberrant sequences. As a consequence, the full-length read alignment may fail. To avoid this problem, seeds of length , derived from the 5′-ends of the reads, are aligned instead. (B) Left: first, we constructed a minimal reference, i.e. the smallest possible region of the reference sequence possibly containing the reconstructed fragment. The construction requires the extension of the reference sequence in correct orientation, starting at the seed alignments, to a total length equal to where corresponds to length of the fragment sequence. The two extensions are ultimately joined to form the minimal reference. Right: next, for an exact identification of the breakpoints a global alignment of the reconstructed fragment sequence and the minimal reference is performed using the Needleman-Wunsch algorithm (see Materials and Methods). The gapped alignment is subsequently used to identify the breakpoint positions corresponding to the start and end position of the gap. (C) Schematic representation of an insertional duplication with inversion of the upstream inserted sequence (blue). Sequencing and processing of the two fragments (frag1 and frag2 in orange and red, respectively) spanning the boundaries of the aberrant region lead to the identification of the insertion site (bp 1) and the two virtual breakpoints (bp 2 and bp 3). The dashed lines connecting the reads of a pair derived from the fragments establish particular connections (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087090#pone-0087090-t003" target="_blank">Table 3</a>) between different breakpoints resulting in an SV-type specific signature on the reference genome. In this example frag1 connects bp 1 and bp 3, approaching both breakpoints from the left. Similarly, frag2 establishes a connection between bp 1 and bp 2, approaching either genomic coordinate from the right. The connections formed by the read pairs can be represented explicitly by introducing directed edges between the different breakpoints. (D) Upper: schematic representation of the discordant graph representing all identified SVs. The example in the inset is similar to C. Lower: the prototype graph for the SV outlined in C. The graph structure represents the signature resulting from the duplication and inversion of the region between bp 2 and bp 3 followed by an upstream insertion at bp 1. Dashed lines highlight an isomorphism between the vertices of the prototype graph and the SV-representing subgraph, since the existence of an edge between two vertices in implies the existence of the same edge connecting the transformed vertices in .</p

Performance comparison on simulated SVs.

<p>Summary of the recall [%] achieved by DSVD, DELLY, BreakDancer, CLEVER and Pindel on different SV types (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087090#pone.0087090.s017" target="_blank">Text S1</a>). The coverages specified during the read simulations are indicated in the legend. intrachr  =  intra chromosomal; interchr  =  inter chromosomal; down  =  downstream; up  =  upstream; no inv  =  no inversion; inv  =  inversion.</p