Breakpoint analysis of balanced chromosome rearrangements by next-generation paired-end sequencing

Abstract

Characterisation of breakpoints in disease-associated balanced chromosome rearrangements (DBCRs), which disrupt or inactivate specific genes, has facilitated the molecular elucidation of a wide variety of genetic disorders. However, conventional methods for mapping chromosome breakpoints, such as in situ hybridisation with fluorescent dye-labelled bacterial artificial chromosome clones (BAC-FISH), are laborious, time consuming and often with insufficient resolution to unequivocally identify the disrupted gene. By combining DNA array hybridisation with chromosome sorting, the efficiency of breakpoint mapping has dramatically improved. However, this can only be applied when the physical properties of the derivative chromosomes allow them to be flow sorted. To characterise the breakpoints in all types of balanced chromosome rearrangements more efficiently and more accurately, we performed massively parallel sequencing using Illumina 1G analyser and ABI SOLiD systems to generate short sequencing reads from both ends of DNA fragments. We applied this method to four different DBCRs, including two reciprocal translocations and two inversions. By identifying read pairs spanning the breakpoints, we were able to map the breakpoints to a region of a few hundred base pairs that could be confirmed by subsequent PCR amplification and Sanger sequencing of the junction fragments. Our results show the feasibility of paired-end sequencing of systematic breakpoint mapping and gene finding in patients with disease-associated chromosome rearrangements.

Figure 1

Ideograms of the patients with breakpoint regions indicated by arrows. (a) Case1. (b) Case 2. (c) Case 3. (d) Case 4.

Figure 2

Junction fragment sequences of cases 1 (a), 2 (b), 3 (c) and 4 (d). The reference sequences are labelled in italic and normal capital characters, respectively. Deleted sequences are underlined. Inserted sequences and single nucleotide variations found on junction fragments are marked by lower case letters and bold capital characters, respectively. Chr, chromosome; Der, derivative chromosome. Junc, junction fragment; 5C, centromeric breakpoint of inv(5); 5T, telomeric breakpoint of inv(5).

Figure 3

Chromosome breakpoints (marked by arrows) and disrupted genes. (a) Case 1, the breakpoint on chromosome 17 disrupts USP43. (b) Case 2, the chromosome 13 breakpoints disrupted a 5′ extended ELF1 isoform. (c) Case 4, the proximal/centromeric breakpoint disrupted RHOBTB3.