Location of Balanced Chromosome-Translocation Breakpoints by Long-Read Sequencing on the Oxford Nanopore Platform

Abstract

Genomic structural variants, including translocations, inversions, insertions, deletions, and duplications, are challenging to be reliably detected by traditional genomic technologies. In particular, balanced translocations and inversions can neither be identified by microarrays since they do not alter chromosome copy numbers, nor by short-read sequencing because of the unmappability of short reads against repetitive genomic regions. The precise localization of breakpoints is vital for exploring genetic causes in patients with balanced translocations or inversions. Long-read sequencing techniques may detect these structural variants in a more direct, efficient, and accurate manner. Here, we performed whole-genome, long-read sequencing using the Oxford Nanopore GridION sequencer to detect breakpoints in six balanced chromosome translocation carriers and one inversion carrier. The results showed that all the breakpoints were consistent with the karyotype results with only ~10× coverage. Polymerase chain reaction (PCR) and Sanger sequencing confirmed 8 out of 14 breakpoints; however, other breakpoint loci were slightly missed since they were either in highly repetitive regions or pericentromeric regions. Some of the breakpoints interrupted normal gene structure, and in other cases, micro-deletions/insertions were found just next to the breakpoints. We also detected haplotypes around the breakpoint regions. Our results suggest that long-read, whole-genome sequencing is an ideal strategy for precisely localizing translocation breakpoints and providing haplotype information, which is essential for medical genetics and preimplantation genetic testing.

Keywords: Oxford Nanopore Technologies; balanced translocation; long-read sequencing; preimplantation genetic testing; structural variants.

Figure 1

Quality-control analysis of the long-read sequencing data obtained using the Oxford Nanopore platform. (A) The median identity of the sequencing data against the reference genome was approximately 85% for all samples. (B) For all samples, the mean lengths were 12.3–16.3 kilobases, and the read N50 values were 15.3–20.5 kilobases. (C) The overall strategy for breakpoint analysis.

Figure 2

Balanced translocation by sequencing and karyotyping for subject DM17A2237. (A) Read mapping of the breakpoints for the balanced translocations. DNA fragments were compared to the reference human genome (GRCh37/hg19), and the breakpoints were shown in IGV. Twenty reads adjacent to the breakpoint were obtained. (B) Karyotype of carrier DM17A2237. Karyotype analysis was determined from G-banding analysis, following a standard protocol. The karyotype result revealed an approximate region where the breakpoint occurred. (C) PCR analysis and Sanger sequencing for validating the breakpoints. An ethidium bromide-stained agarose gel was showing the presence of two new bands created by the rearrangement of chromosomal segments at breakpoints (BP1 and BP2). BP, breakpoint; C, control; M, marker. Primer information is available in Table S1 .

Figure 3

Long-read sequencing enabled haplotype detection around the translocation breakpoints in sample DM17A2237. Using the breakpoints as anchoring markers, we obtained 2-megabase sequences on either side of the breakpoints. Through SNP calling and the MarginPhase tool, we phased the haplotypes around the breakpoints in chr18 (A) and chr21 (B). Reads around breakpoints were shown in IGV (bottom panel) and regions in red box were enlarged (top panel). Capital letters represent accurate sequencing information, whereas lowercase letters represent fuzzy base information.