Amplification and thrifty single-molecule sequencing of recurrent somatic structural variations

Abstract

Deletion of tumor-suppressor genes as well as other genomic rearrangements pervade cancer genomes across numerous types of solid tumor and hematologic malignancies. However, even for a specific rearrangement, the breakpoints may vary between individuals, such as the recurrent CDKN2A deletion. Characterizing the exact breakpoints for structural variants (SVs) is useful for designating patient-specific tumor biomarkers. We propose AmBre (Amplification of Breakpoints), a method to target SV breakpoints occurring in samples composed of heterogeneous tumor and germline DNA. Additionally, AmBre validates SVs called by whole-exome/genome sequencing and hybridization arrays. AmBre involves a PCR-based approach to amplify the DNA segment containing an SV's breakpoint and then confirms breakpoints using sequencing by Pacific Biosciences RS. To amplify breakpoints with PCR, primers tiling specified target regions are carefully selected with a simulated annealing algorithm to minimize off-target amplification and maximize efficiency at capturing all possible breakpoints within the target regions. To confirm correct amplification and obtain breakpoints, PCR amplicons are combined without barcoding and simultaneously long-read sequenced using a single SMRT cell. Our algorithm efficiently separates reads based on breakpoints. Each read group supporting the same breakpoint corresponds with an amplicon and a consensus amplicon sequence is called. AmBre was used to discover CDKN2A deletion breakpoints in cancer cell lines: A549, CEM, Detroit562, MOLT4, MCF7, and T98G. Also, we successfully assayed RUNX1-RUNX1T1 reciprocal translocations by finding both breakpoints in the Kasumi-1 cell line. AmBre successfully targets SVs where DNA harboring the breakpoints are present in 1:1000 mixtures.

Figure 1.

PAMP tiling design for capture of CDKN2A deletions. CDKN2A upstream and downstream breakpoint regions are defined on a germline genome, blue and red lines, respectively. Tiled forward primers (blue arrows) and reverse primers (red arrows) are spaced ≈1 kb apart (width of hashed boxes; not to scale with reference). Overlap of blue box and red box on tumor DNA indicates that a forward and reverse primer pair is <2 kb apart and will lead to amplification of tumor DNA harboring CDKN2A deletion breakpoints.

Figure 2.

AmBre pipeline with primer designing and PacBio long fragment sequence analysis.

Figure 3.

Designing AMBRE-68. (A) Candidate primers are uniformly distributed in CDKN2A locus, suggesting that good primer designs are possible. AmBre-design is tasked to capture CDKN2A deletion upstream and downstream breakpoints in regions chr9: 21,730,000–21,965,000 and chr9: 21,975,000–22,129,000 (GRCh37 coordinates), respectively. (B) Final low-cost 68-primer design to capture CDKN2A deletions in 380-kb breakpoint region. The solution has a 97.6% and a 99.7% coverage of breakpoint regions. The fraction of break pairs captured by the design (resulting in amplicon length <13 kb) is 99.99%.

Figure 4.

Aggregates of breakpoints from each PacBio fragments after sweep line clustering. Target amplicons are strongly supported by fragments and breakpoints are well separated. Only breakpoints with L < 1 kb are displayed for inset boxes. The height of each cluster corresponds with number of fragments supporting the breakpoint (depth of breakpoint coverage).

Figure 5.

Subsampling of nine primers from the complete AMBRE-68 tiling design results in clean amplification of CDKN2A loss DNA fragments in six cell lines. (From left to right) Lanes contain 1 kb of Plus GeneRuler DNA ladder, PCR products from samples A549 (2.2 kb), CEM (5.8 kb), MCF7 (3.6 kb), MOLT4 (6.8 kb), T98G (7.5 kb), HEK, and water. The expected lengths of each amplicon according to AMBRE-68 design are listed in parentheses. HEK cells (no CDKN2A deletion) and H₂O are negative controls.

Figure 6.

Characterizing RUNX1–RUNX1T1 balanced translocation in Kasumi-1. Lanes 1, 2, 4, 6, and 8 contain 1 kb of Plus GeneRuler DNA ladder, PCR products from Kasumi-1 Der8 with all 28 primers (3.5 kb), 14 primer FE ∪ RO (3.5 kb), 14 primer FO ∪ RO (6.8 kb), and 14 primer FO ∪ RE (10.1 kb). Lanes 3, 5, 7, and 9 contain matching water controls, which show no contamination. Lanes 10, 12, 14, and 16 contain PCR products from Kasumi-1 Der21 with all 29 primers (2.7 kb), 15 primer FO ∪ RO (2.7 kb), 15 primer FE ∪ RO (6.1 kb), and 14 FE ∪ RE (8.1 kb). The gel was loaded with 2 μL for lanes 2–5 and 10–13, and 4 μL for remaining volumes. Reactions with shorter amplicons amplified extremely well, and lesser volumes were used for visualization on the gel. The expected amplicon lengths according to the Der8 and Der21 design are listed in parentheses.

Figure 7.

(A) Fragment-segmentation example for local alignments 1, 2, 3, and 4 along a PacBio fragment. (B) Triangle representation of adjacent alignments 1, 2, and 3 on G × G plane.