Chromosomal Junction Detection from Whole-Genome Sequencing on Formalin-Fixed, Paraffin-Embedded Tumors

Stephen Murphy, James Smadbeck, Bruce Eckloff, Yean Lee, Sarah Johnson, Giannoula Karagouga, Vishnu Serla, Anurag Sharma, Robert Sikkink, Jesse Voss, Faye Harris, Janet Schaefer Kline, Farhad Kosari, Andrew Feldman, Eric Wieben, Marie Christine Aubry, Benjamin Kipp, Jin Jen, John Cheville, George Vasmatzis

Research output: Contribution to journalArticlepeer-review


DNA junctions (DNAJs) frequently impact clinically relevant genes in tumors and are important for diagnostic and therapeutic purposes. Although routinely screened through fluorescence in situ hybridization assays, such testing only allows the interrogation of single-gene regions or known fusion partners. Comprehensive assessment of DNAJs present across the entire genome can only be determined from whole-genome sequencing. Structural variance analysis from whole-genome paired-end sequencing data is, however, frequently restricted to copy number changes without DNAJ detection. Through optimized whole-genome sequencing and specialized bioinformatics algorithms, complete structural variance analysis is reported, including DNAJs, from formalin-fixed DNA. Selective library assembly from larger fragments (>500 bp) and economical sequencing depths (300 to 400 million reads) provide representative genomic coverage profiles and increased allelic coverage to levels compatible with DNAJ calling (40× to 60×). Although consistently fragmented, more recently formalin-fixed, specimens (<2 years’ storage) revealed consistent populations of larger DNA fragments. Optimized bioinformatics efficiently detected >90% of DNAJs in two prostate tumors (approximately 60% tumor) previously analyzed by mate-pair sequencing on fresh frozen tissue, with evidence of at least one spanning-read in 99% of DNAJs. Rigorous masking with data from unrelated formalin-fixed tissue progressively eliminated many false-positive DNAJs, without loss of true positives, resulting in low numbers of false-positive passing current filters. This methodology enables more comprehensive clinical genomics testing on formalin-fixed clinical specimens.

Original languageEnglish (US)
Pages (from-to)375-388
Number of pages14
JournalJournal of Molecular Diagnostics
Issue number4
StatePublished - Apr 2021

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine


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