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Defining cat mitogenome variation and accounting for numts via multiplex amplification and Nanopore sequencing
Emily C Patterson1, Gurdeep Matharu Lall1, Rita Neumann1
1Department of Genetics & Genome Biology, University of Leicester, University Road, Leicester LE1 7RH UK.
Forensic Science International. Genetics
|October 11, 2023
Summary
Forensic DNA analysis of cat hair is improved using a new multiplex sequencing method that analyzes the whole mitochondrial genome. This approach increases genetic diversity and reduces random match probabilities for reliable cat identification.
Area of Science:
- Forensic Science
- Genetics
- Molecular Biology
Background:
- Cat hair DNA is valuable forensic evidence, but low mitochondrial DNA (mtDNA) diversity limits its discriminatory power.
- Current methods sequencing only a control region segment yield limited genetic variation.
- Previous whole mitogenome sequencing in cats increased diversity but required a long-amplicon approach.
Purpose of the Study:
- To develop a multiplex system for amplifying and sequencing the entire cat mitogenome from hair for forensic applications.
- To assess the impact of nuclear mitochondrial insertion sequences (numts) on mitogenome analysis.
- To demonstrate the forensic utility of the developed method in a real-world case.
Main Methods:
- A multiplex system was designed to amplify the cat mitogenome into 60 overlapping amplicons.
- Nanopore sequencing was used to analyze the amplified DNA from cat hair samples.
- Long-amplicon sequencing and analysis of published genome data were employed to investigate numt variation.
Main Results:
- Multiplex sequencing of hair DNA accurately mirrored whole mitogenome data from blood.
- Co-amplification with feline nuclear mitochondrial insertion sequences (numts) was observed in blood DNA, revealing variability in numt structure and sequence.
- The method successfully matched cat hairs to skeletal remains, significantly reducing random match probability compared to control region sequencing.
Conclusions:
- The developed multiplex and Nanopore sequencing approach provides a reliable and highly discriminating source of forensic genetic evidence from cat hair, especially when nuclear DNA is scarce.
- Understanding and accounting for numt variation is crucial for accurate interpretation of mitogenome data, particularly in degraded samples.
- This method enhances the forensic value of cat hair by leveraging increased genetic diversity from whole mitogenome analysis.
