High-resolution mapping of reciprocal translocation breakpoints using long-read sequencing

Abstract

Long-read nanopore sequencing enables direct high-resolution breakpoint mapping on balanced carriers of reciprocal translocation. The mean sequencing depth on the translocated chromosomes to achieve accurate mapping of breakpoints ranged from 2.5-fold to 6.2-fold. To speed up determination of the breakpoints from long-read sequencing data, alignment reads on the translocated chromosomes were extracted before piped into NanoSV. Checking the position of breakpoints on Interactive Genomics Viewer (IGV) was crucial to successful design of breakpoint PCR primers, especially when large deletion was involved at the breakpoints. •Long-read sequencing enables accurate breakpoint mapping with base-pair resolution•Splitting bam files by translocated chromosomes drastically speeded up the breakpoint determination•IGV helps to identify the breakpoint positions and facilitate the design of breakpoint PCR primers.

Keywords: Breakpoint; Nanopore sequencing; PGT-SR; Translocation breakpoint mapping using nanopore sequencing.

Graphical abstract

Fig. 1

Aligned reads shown on IGV and Sanger sequencing results of derivative chromosomes of sample #100238. (A) Aligned reads on chromosome 2. (B) Aligned reads on chromosome 10. (C–D) Sanger sequencing results on der(2) and der (10) respectively, showing highly concordant results with IGV views. Red arrow indicates the breakpoints predicted by NanoSV.

Fig. 2

Aligned reads shown on IGV and breakpoint PCR results of sample #100648. (A) Aligned reads on chromosome 8. (B) Aligned reads on chromosome 10. Red arrow indicates breakpoints predicted by NanoSV. (C) First breakpoint PCR results. Primers on chromosome 10 were designed (10 F and 10R) solely based on the predicted breakpoint (chr10:128,448,174). (D) Second breakpoint PCR results. Primer were designed (10 F2 and 10R2) based on the chimeric read (depicted in blue box) located at the 3′ end of predicted breakpoint, visualized on IGV. N: noncarrier; C: translocation carrier; B: negative control; M: DNA molecular weight marker.