Preparing for Shotgun Sequencing in Forensic Genetics—A Workflow for Analysis of Monozygotic Twins

BACKGROUND/OBJECTIVES: Identifying the true perpetrator among monozygotic (MZ) twins has long posed a challenge in forensic genetics and for police investigations because MZ twins are likely identical in their short tandem repeat (STR) profile. In this study, we propose a workflow to address this issue, anchored in a case study involving MZ twins. METHODS: The workflow includes shotgun sequencing of reference samples from one twin and trace samples, followed by a targeted amplicon-based approach (AmpliSeq™ Custom Panel) to validate the identified variants. RESULTS: Biological traces from the crime scene, identified as blood, were available for analysis along with reference material from one twin, collected as a buccal swab. The samples underwent shotgun sequencing, and the resulting reads were aligned to the human reference genome. The sequencing yielded 2.91 and 2.85 billion mapped positions (corresponding to a breadth of coverage of 91% and 89%), with mean depths of coverage of 39.7× and 25.8× for the trace and reference samples, respectively. To minimise the risk of detecting somatic variants in the two different tissues, a stringent heterozygosity balance threshold (Hb: 0.4-0.6) was applied, and 2,047,077 single nucleotide variants (SNVs) were identified, of which 28 showed inconsistencies between the trace and reference samples. These candidate variants were subjected to validation using an AmpliSeq™ Custom Panel. CONCLUSIONS: No detectable SNV differences were observed between the reference and trace samples, and it was not possible to determine whether the trace originated from the reference twin or from his monozygotic co-twin.