Department of Genetics & Genome Biology, University of Leicester, University Road, Leicester LE1 7RH UK.
IDEXX Laboratories Italia S.r.l., Via Guglielmo Silva, 36-20149 Milano (MI), Italy.
Forensic Sci Int Genet. 2023 Nov;67:102944. doi: 10.1016/j.fsigen.2023.102944. Epub 2023 Oct 6.
Hair shed by domestic cats is a potentially useful source of forensic evidence. Analysable hair DNA is predominantly mitochondrial, but the recent domestication history of cats means that mtDNA diversity is low. A 402-bp control region segment is usually sequenced, defining only a small number of distinct haplotypes in populations. Previously, we used a long-amplicon approach to sequence whole mitogenomes in a sample of blood DNAs from 119 UK cats, greatly increasing observed diversity and reducing random match probabilities. To exploit this variation for forensic analysis, we here describe a multiplex system that amplifies the cat mitogenome in 60 overlapping amplicons of mean length 360 bp, followed by Nanopore sequencing. Variants detected in multiplex sequence data from unrooted hair completely mirror those from long-amplicon data from blood from the same individuals. However, applying the multiplex to matched blood DNA reveals additional sequence variants which derive from the major feline nuclear mitochondrial insertion sequence (numt), which covers 7.9 kb of the 17-kb mitogenome and exists in multiple tandem copies. We use long-amplicon Nanopore sequencing to investigate numt variation in a set of cats, together with an analysis of published genome sequences, and show that numt arrays are variable in both structure and sequence, thus providing a potential source of uncertainty when nuclear DNA predominates in a sample. Forensic application of the multiplex was demonstrated by matching hairs from a cat with skeletal remains from its putative mother, both of which shared a globally common haplotype at the control region. The random match probability in this case with the CR 402-bp segment was 0.21 and this decreased to 0.03 when considering the whole mitogenome. The developed multiplex and sequencing approach, when applied to cat hair where nuclear DNA is scarce, can provide a reliable and highly discriminating source of forensic genetic evidence from a single hair. The confounding effect of numt co-amplification in degraded samples where mixed sequences are observed can be mitigated by variant phasing, and by comparison with numt sequence diversity data, such as those presented here.
家猫的脱落毛发是一种有潜在用途的法医证据来源。可分析的毛发 DNA 主要是线粒体 DNA，但由于猫最近的驯化历史，mtDNA 多样性较低。通常会对 402bp 的控制区片段进行测序，这仅在种群中定义了少数不同的单倍型。此前，我们使用长扩增子方法对来自 119 只英国猫的血液 DNA 样本中的整个线粒体基因组进行测序，极大地增加了观察到的多样性并降低了随机匹配概率。为了将这种变异用于法医分析，我们在这里描述了一种扩增子系统，该系统可在 60 个平均长度为 360bp 的重叠扩增子中扩增猫的线粒体基因组，然后进行纳米孔测序。在未生根毛发的多路复用序列数据中检测到的变体完全反映了来自同一个体血液的长扩增子数据中的变体。然而，将多路复用应用于匹配的血液 DNA 会揭示出源自主要的猫核线粒体插入序列（numt）的额外序列变体，该序列覆盖了 17kb 线粒体基因组的 7.9kb，并且存在多个串联副本。我们使用长扩增子纳米孔测序来研究一组猫中的 numt 变异，以及对已发表基因组序列的分析，并表明 numt 阵列在结构和序列上都是可变的，因此当核 DNA 在样本中占主导地位时，可能会导致不确定性。通过匹配来自疑似母亲的骨骼遗骸中的猫毛，展示了多路复用的法医应用，两者在控制区都共享了全球共同的单倍型。在这种情况下，使用 CR 402bp 片段的随机匹配概率为 0.21，当考虑整个线粒体基因组时，该概率降低至 0.03。在核 DNA 稀少的猫毛中应用开发的多路复用和测序方法，可以提供一种可靠且高度区分的法医遗传证据来源，仅需一根毛发。在观察到混合序列的降解样本中，numt 共扩增的混杂效应可以通过变体相位来缓解，并通过与 numt 序列多样性数据（如这里提供的）进行比较来缓解。
