Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite

Patterson, Emily J; Lall, Gurdeep Matharu; Neumann, Rita; Ottolini, Barbara; Batini, Chiara; Sacchini, Federico; Foster, Aiden P; Wetton, Jon H.; Jobling, Mark A.

doi:10.1101/2023.06.13.544779

Cited by 1 publication

(11 citation statements)

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“…Primer pairs were designed to cover the cat mitogenome in sixty overlapping amplicons of average length ~360 bp. Design was based on an alignment of 119 domestic cat mitogenomes (accession numbers OR085738 -OR085764; OR077766 -OR077783; OR077785 -OR077857 [20]), with 92 complete sequences obtained through Nanopore sequencing and 26 through Illumina sequencing, plus the domestic cat reference mitogenome (NC_001700 [12]). Primers were designed with the aid of AliView (v1.26) [22] (Table S1), allowing haplotype-specific SNPs to be taken into account either by primer positioning or the incorporation of degenerate nucleotides.…”

Section: Multiplex Design Amplification and Sequencing Of Mtdnamentioning

confidence: 99%

“…(a) Structure of cat mtDNA based on the NC_001700 reference sequence [12], showing genes and the extent of the large numt. Primers for the two long overlapping amplicons used for sequencing [20] are indicated by short coloured arrows. The control region includes the positions of the RS2 and RS3 repeat arrays, and the 402-bp segment usually sequenced; its 5´-3ó rientation is according to [44].…”

Section: Introductionmentioning

confidence: 99%

“…Note that each primer site is expected to exist within each 7.9-kb repeat unit, and (with the exception of amplicon 2) the resulting amplicon spans two adjacent repeat units in the array. (c) Median-joining network based on variants within the control region within 119 cat mitogenomes [20]. Circles represent haplotypes, with area proportional to sample size, and lines between haplotypes represent mutational steps as shown in the key.…”

Section: Introductionmentioning

confidence: 99%

“…To provide a higher degree of discrimination, a more powerful method than CR sequencing is necessary. In a previous study, we approached this by using Nanopore and Illumina technology to sequence the whole mitogenomes of 119 cats, using a long-amplicon approach to avoid the problem of the major cat numt [20]. Excluding the RS2 and RS3 repetitive regions, this allowed the extensive subdivision of mitotypes (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…1d), and a near order-of-magnitude reduction of random match probability (RMP) from 0.16 to 0.018. Nanopore sequencing also allowed variation within the RS2 repeat array to be defined, revealing complex and dynamic minisatellite-like structures with both repeat number and repeat sequence variation, that further increase the diversity of cat mitogenomes [20].…”

Section: Introductionmentioning

confidence: 99%

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Defining cat mitogenome variation and accounting for numts via multiplex amplification and Nanopore sequencing

Patterson

Lall

Neumann

et al. 2023

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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 mitotypes 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 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 test was demonstrated by matching hairs from a cat with skeletal remains from its putative mother, both of which shared a globally common mitotype at the control region. The random match probability (RMP) 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. 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.

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Section: Multiplex Design Amplification and Sequencing Of Mtdnamentioning

confidence: 99%