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Resolving highly complex rearrangements of genomic architecture using long PromethION reads poster


Date: 28th November 2018

Genomic disorders are diseases that result from chromosomal rearrangements, rather than from nucleotide-scale changes, and lead to the loss or gain of chromosomal material, or to inversions.

Fig. 1 Non-allelic homologous recombination a) and b) mechanisms c) examples of disorders

Deletion, duplication and inversion of dosage-sensitive genes

Incorrect pairing of direct homologous repeats during meiosis can result in non-allelic recombination. This reciprocal event leads to one gamete with duplication of the intervening sequence and one with deletion (Fig. 1a). Recombination between inverted repeats results in inversion of the intervening sequence, which can disrupt gene function by inverting exons, or by disconnecting coding and regulatory regions (Fig. 1b). If affected sequences contain dosage-sensitive genes, the effect on the offspring can range from mild to devastating (Fig. 1c). Long reads are useful for precise characterisation of breakpoints and assembly of repetitive regions.

Fig. 2 Cri du Chat syndrome a) genetics b) relative copy number revealed by read depth

Detecting the Cri du chat deletion on the short arm of chromosome 5

Cri du chat syndrome is a rare disorder, affecting 1 in 50,000 live births, with symptoms including severe cognitive, speech and motor difficulties, and behavioural problems. The disorder results from a partial deletion of the short arm of chromosome 5, usually from the paternal chromosome (Fig. 2a). To detect the deletion we sequenced gDNA from a Cri du Chat cell line. We aligned reads to the reference genome with ngmlr and used Mosdepth to measure depth in 100 kb bins. Raw depth was median normalised and processed using an HMM to assign an integer ploidy state to each bin. The deletion is clearly visible, along with cell-line artefacts (Fig 2b).

Fig. 3 Duplication-inverted triplication-duplication rearrangement a) mechanism b) analysis of PromethION sequence data: absence of heterozygosity; copy number changes; structural variant calls

Using long whole-genome PromethION data to fully characterise a duplication-inverted triplication-duplication rearrangement on the long arm of chromosome 14

The duplication-inverted triplication-duplication structure (Fig. 3a) is a class of complex genomic rearrangement that can lead to severe phenotypic consequences. Just two breakpoints can give rise to both duplicated and triplicated regions. To determine the mechanism that gave rise to the rearrangement it is necessary to i) distinguish between duplicated and triplicated regions, ii) identify breakpoint junctions and iii) identify any absence of heterozygosity (AOH) created by the event. Here we analysed a sample where the rearranged locus had previously been identified using array-CGH, but the method gave insufficient resolution to identify the duplicated regions and thus the breakpoints could not be ascertained. The sample was sequenced to a mean coverage of 16x on a PromethION and we obtained an aligned read N50 of 15 kb. Reads were aligned against human reference genome hg38 using ngmlr and the resulting alignments were analysed by Sniffles to detect structural variants. Copy number was estimated by comparing read depth at each position with that of four control samples and applying a correction factor for different amounts sequenced per sample. Sniffles detected 26,036 bp and 2,263 bp inversions (the latter as an inverted duplication) at the centromeric and telomeric ends of the locus respectively. In both cases one or more reads spanned the entire length of the duplicated region (visible as reads where the alignments from both strands overlap). We were able to determine both breakpoints and these were confirmed by capillary sequencing. To identify AOH we called SNPs using bcftools and compared the density of heterozygous SNPs in the target sample against the controls – a moving average of 10 bins is plotted in the top panel of Fig. 3b.

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