Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

10.1371/journal.pone.0046152PLoS ONE79

dPET cluster characteristics.

<p>(A) PET mapping overlap scheme based on the same number of PETs. 10 kb library dPETs have a higher chance than 1 kb library dPETs to cross the same breakpoints. (B) One common deletion identified in K562 by 1 kb, 10 kb and 20 kb libraries. Red track represents the coverage of the cPETs. The dPET cluster count in each library was 6, 51 and 95, respectively. The genomic PCR and Sanger sequencing confirmed the presence of the deletion and located the breakpoint positions to chr10:126,631,456 and chr10:126,720,709 (hg18). The differences between breakpoint position predicted by DNA-PET and PCR were 387 bp and 238 bp for the 1 kb library; 333 bp and 36 bp for the 10 kb library, and 482 bp and 37 bp in 20 kb library. (C) Cluster count correlation of the same set of SVs identified by both 1 kb and 10 kb libraries in the three genomes (left panel), and 10 kb and 20 kb libraries in K562 (right panel). The black line represents the trendline. The rearrangement between chromosomes 9 and 22 creating the CML causing <i>BCR-ABL1</i> fusion gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046152#pone.0046152-Groffen1" target="_blank">[23]</a> was identified by the largest clusters of 692 dPETs in the 10 kb library and 2,106 dPETs in the 20 kb library (red arrow head). (D) Length distribution of 5′ and 3′ anchor regions (regions in which the tags of dPET clusters are mapping) in 1 kb, 10 kb and 20 kb libraries of K562. The 10 kb library showed a more even length distribution of 5′ and 3′ anchor regions which suggested more balanced mapping characteristics around breakpoints for 10 kb libraries.</p

Comparison of SVs identified by 1 kb and 10 kb libraries in the three genomes.

<p>(A–B) Number of SVs identified in 1 kb libraries compared to the number of SVs identified in 10 kb libraries. Top, Venn diagrams showing the respective numbers of SVs in each library type and the overlap of SVs. Percentages in parentheses represent SVs identified by 1 kb or 10 kb libraries in all three genomes. Bottom, number of SVs (y-axis) of the indicated SV categories was shown for the specific span sizes (x-axis). (C) Breakpoints confirmed by PCR and Sanger sequencing and their resolution (defined as the distance in bp between the predicted and actual breakpoints). (D) A 10 kb library specific deletion in MCF-7. The genomic PCR and Sanger sequencing confirmed left and right breakpoints are chr9:22,486,527 and chr9:22,494,345, respectively (hg18). The resolution of the left and right sides of the deletion are 906 bp and 4,683 bp, respectively. Repetitive sequence which does not allow unambiguous mapping covers the entire 4,683 bp region. The repetitive sequence could not be spanned by the 1 kb library preventing the identification of this deletion. (E) A deletion in MCF-7 identified by both 10 kb and 1 kb libraries. The left and right breakpoints confirmed by genomic PCR and Sanger sequencing are at chr3:6,625,155 and chr3:6,629,779, respectively (hg18). Based on the 10 kb library predicted breakpoints, the resolution on the left and right sides of the deletion are 973 bp and 2,216 bp, respectively. The tags of the 1 kb library mapped to the gap (in orange) between the repetitive sequences and allowed the identification of this deletion.</p

Comprehensive long-span paired-end-tag mapping reveals characteristic patterns of structural variations in epithelial cancer genomes

Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long-span paired-end-tag (PET) sequencing approach using 10-Kb genomic DNA inserts to study human genome structural variations (SVs). The use of a 10-Kb insert size allows the identification of breakpoints within repetitive or homology-containing regions of a few kilobases in size and results in a higher physical coverage compared with small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and two noncancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germ-line SVs, whereas tandem duplications, unpaired inversions, interchromosomal translocations, and complex rearrangements are over-represented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA–PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures that are compatible with breakage-fusion-bridge cycles, and we discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers

SV identification based on the mapping pattern of dPET clusters.

<p>The dark red and pink arrows represent the 5′ and 3′ anchor regions of the dPET cluster, respectively. Black, white and blue horizontal lines represent chromosome segments. The red track represents the coverage of cPETs. The dotted lines indicate the connections between the two dPET clusters. The sub-types of insertions are as follows: (1) Intra-chromosomal direct forward insertion. (2) Intra-chromosomal direct backward insertion. (3) Intra-chromosomal inverted forward insertion. (4) Intra-chromosomal inverted backward insertion. (5) Deletion plus intra-chromosomal direct forward insertion. (6) Deletion plus intra-chromosomal inverted forward insertion. (7) Inter-chromosomal direct insertion. (8) Inter chromosome inverted insertion.</p

Reconstruction of the <i>BCR-ABL1</i> amplicon of K562.

<p>(A) Concordant tag distributions representing copy number are shown for amplified genomic regions (top, green track). Genomic segments between predicted breakpoints are indicated by colored arrows and dPET clusters with cluster sizes greater than 35 of predicted somatic rearrangements are represented by horizontal lines flanked by dark red and pink arrows indicating 5′ and 3′ anchor regions (middle). Small to large dPET clusters are arranged from top to bottom. Cluster sizes are indicated. High dPET cluster size of the CML causing <i>BCR-ABL1</i> translocation suggests that the rearrangement occurred early and that it has subsequently been amplified. Fusion points I–III correspond to panels C–D. (B) Fluorescence <i>in situ</i> hybridization (FISH) of <i>BCR-ABL1</i> rearrangement (fusion point I with cluster size 692). Yellow spots represent fusion signals and illustrate the amplification of <i>BCR-ABL1</i>. (C) FISH analysis of metaphase chromosomes of three high copy fusion points: I) probes used in B show fusion signals on two marker chromosomes and on chromosome 2q and normal localization on both rearranged chromosomes 9 and normal chromosome 22; the fusion on chromosome 2 has not been identified by DNA-PET most likely due to low sequence complexity at the break point or complex rearrangements, II) probes spanning the fusion point II (cluster size 259) show fusion signals on the same marker chromosomes and normal localization on both normal and rearranged chromosomes 9 and 13, III) probes spanning fusion point III (cluster size 218) show fusion signals on the same marker chromosomes and normal localization on both normal chromosome 22 and rearranged chromosomes 9. (D) Contigs (indicated by boxes) which were covered by PET mapping were concatenated by fusion-point-guided-concatenation method. The length of a contig is represented by the length of the box. Because of the size difference between chromosomes 1, 3, 9, 13, and 22, the length of chromosome 22 is represented by the length of contig/10,000 while the lengths of chromosomes 1, 3, 9, and 13 are represented by the length of contig/100,000. Any value less than 0.1 is rounded to 0.1; any value larger than 6 is rounded to 6. The thickness of borders of each contig represents the coverage (copy number). Red dashed edges represent dPET edges, while black bold edges represent cPET edges. The thickness of dPET edges represents the size of the corresponding dPET cluster. cPET edges have uniform thickness. Arrow heads pointing towards a contig indicate connections with the lower coordinates, arrow heads pointing away from a contig indicate connections with the higher coordinates.</p

DNA-PET library construction, sequencing and mapping.

<p>(A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, <i>EcoP15I</i> digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.</p

Statistics of PET sequencing.

1)<p>non-redundant.</p>2)<p>physical coverage.</p>3)<p>concordant PET.</p>4)<p>proportion of cPET to total NR PET.</p>5)<p>discordant PET.</p>6)<p>proportion of dPET to total NR PET.</p>7)<p>number of dPET involved in the dPET clusters with cluster count ≥3.</p