Resolving Breakpoints of Chromosomal Rearrangements at the Nucleotide Level Using Sanger Sequencing

Novel cytogenetic tools are increasingly based on genome sequencing for detecting chromosomal abnormalities. Different sequence-based techniques optimized for diagnosis of structural variants can be useful for narrowing down the localization of breakpoints of chromosomal abnormalities, but do not offer nucleotide resolution of breakpoints for proper interpretation of gene disruption. This protocol presents the characterization of structural variants at nucleotide resolution using Sanger sequencing after low-pass large-insert genome sequencing or other long-molecule methods. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Primer design for junction amplification at translocations and inversions

Basic Protocol 2: Amplification of derivative chromosomes using a long-range polymerase

Alternate Protocol: Amplification of derivative chromosomes using a hot-start polymerase

Basic Protocol 3: Preparation of DNA for Sanger sequencing

Basic Protocol 4: Interpretation and reporting of breakpoints based on Sanger sequencing