Germline long insertions in BRCA1/PALB2 exon revealed by PCR-free short-read and long-read whole-genome sequencing (Hybrid poster presentation)

Abstract

<p>Background/Objectives: While to detect structural variants disrupting hereditary breast cancer genes is important, the detection of long insertion (>100 bp) is challenging. <br></p><p>Methods: Short-read PCR-free whole-genome sequencing (WGS) libraries were constructed using KAPA EvoPlus Kit (Roche), sequenced at 2x151 bp on NovaSeq 6000 S1 flow cells (Illumina), followed by alignment using Clara Parabricks v4.0.0 (Nvidia) and insertion detection using INSurVeyor v1.1.2. Long-read PCR-free WGS libraries were constructed using Ligation Sequencing Kit V14, sequenced on PromethION R10.4.1 flow cells (Oxford Nanopore), followed by Dorado v4.2.0 super-accurate basecalling, alignment using minimap2 v2.24 and consensus sequence generation using Flye v2.9. <br></p><p>Results: Under the hereditary breast cancer genetic testing program of Hong Kong Hereditary Breast Cancer Family Registry, we identified germline long insertions from two probands. Proband 1 was a Chinese female bilateral breast cancer patient with positive family history. A heterozygous 318 bp insertion with AluY and target site duplication at PALB2 exon 11 was identified by both short-read and long-read WGS. Proband 2 was a Chinese female early-onset triple-negative breast cancer patient with positive family history. An insertion at BRCA1 exon 24 was suspected by short-read WGS and fully elucidated by long-read WGS as a heterozygous complex deletion of 12 bp and insertion of 12,423 bp. It is among the longest insertions reported in hereditary breast cancer genes as deciphered by WGS. <br></p><p>Conclusion: Detection of the germline insertions enabled better-informed patient management and family member testing.<br></p>