Biology subjects

Proukakis, C.

Publications and source records attributed to Proukakis, C..

6 recordsLinked to original sources

bioRxiv2026

Targeted single-nucleus sequencing of 39,800 neurons reveals extensive low-frequency somatic variants

Recent evidence implicates somatic mutations in disease-associated genes across multiple neurodegenerative disorders, including the identification of two somatic TARDBP variants as a putative cause of frontotemporal lobar degeneration with TDP-43 pathology type C (FTLD-TDP type C). However, these findings remain anecdotal. We performed single-nucleus amplicon sequencing of 39,800 neurons from the superior temporal gyrus of sporadic FTLD-TDP type C patients (34,738 neurons, 52 individuals) and non-demented controls (5,062 neurons, 26 individuals) using the Mission Bio Tapestri platform and investigated somatic variants in TARDBP and other FTLD-TDP-associated genes in a case-control setting. We uncovered an extensive landscape of ultra-low-frequency (<1%) somatic mutations across all analysed genes, including TARDBP. Rare somatic occurrences of known ALS/FTD-associated germline TARDBP variants, including p.N267S and p.M337V, were identified exclusively in patients. Among all targeted genes, TARDBP had the highest proportion of neurons harbouring a somatic variant, and this mutational burden was lower in individuals who died at an older age. Strikingly, the C-terminal domain of TDP-43, where most ALS/FTD germline pathogenic variants reside, exhibited a lower neuronal mutational burden than its other domains. This suggests that neurons harbouring damaging mutations may undergoprogressive loss prior to autopsy, leaving only rare survivors at frequencies too low to detect by bulk or small-scale single-cell approaches. Together, our findings provide an unprecedented resolution into the somatic mutational landscape of post-mitotic neurons in FTLD-TDP type C and reveal a gene-specific layer of neuronal mosaicism that current single-cell WGS studies fail to detect.

genetics↗

bioRxiv2023

Single-cell somatic copy number variants in brain using different amplification methods and reference genomes

The presence of somatic mutations, including copy number variants (CNVs), in the brain is well recognized. Comprehensive study requires single-cell whole genome amplification, with several methods available, prior to sequencing. We compared PicoPLEX with two recent adaptations of multiple displacement amplification (MDA): primary template-directed amplification (PTA) and droplet MDA, across 93 human brain cortical nuclei. We demonstrated different properties for each, with PTA providing the broadest amplification, PicoPLEX the most even, and distinct chimeric profiles. Furthermore, we performed CNV calling on two brains with multiple system atrophy and one control brain using different reference genomes. We found that 38% of brain cells have at least one Mb-scale CNV, with some supported by bulk sequencing or single-cells from other brain regions. Our study highlights the importance of selecting whole genome amplification method and reference genome for CNV calling, while supporting the existence of somatic CNVs in healthy and diseased human brain.

neuroscience↗

bioRxiv2022

Comprehensive Structural Variant Detection: From Mosaic to Population-Level

Long-read Structural Variation (SV) calling remains a challenging but highly accurate way to identify complex genomic alterations. Here, we present Sniffles2, which is faster and more accurate than state-of-the-art SV caller across different coverages, sequencing technologies, and SV types. Furthermore, Sniffles2 solves the problem of family- to population-level SV calling to produce fully genotyped VCF files by introducing a gVCF file concept. Across 11 probands, we accurately identified causative SVs around MECP2, including highly complex alleles with three overlapping SVs. Sniffles2 also enables the detection of mosaic SVs in bulk long-read data. As a result, we successfully identified multiple mosaic SVs across a multiple system atrophy patient brain. The identified SV showed a remarkable diversity within the cingulate cortex, impacting both genes involved in neuron function and repetitive elements. In summary, we demonstrate the utility and versatility of Sniffles2 to identify SVs from the mosaic to population levels.

bioinformatics↗

bioRxiv2019

SVCurator: A Crowdsourcing app to visualize evidence of structural variants for the human genome

A high quality benchmark for small variants encompassing 88 to 90% of the reference genome has been developed for seven Genome in a Bottle (GIAB) reference samples. However a reliable benchmark for large indels and structural variants (SVs) is yet to be defined. In this study, we manually curated 1235 SVs which can ultimately be used to evaluate SV callers or train machine learning models. We developed a crowdsourcing app - SVCurator - to help curators manually review large indels and SVs within the human genome, and report their genotype and size accuracy.\n\nSVCurator is a Python Flask-based web platform that displays images from short, long, and linked read sequencing data from the GIAB Ashkenazi Jewish Trio son [NIST RM 8391/HG002], We asked curators to assign labels describing SV type (deletion or insertion), size accuracy, and genotype for 1235 putative insertions and deletions sampled from different size bins between 20 and 892,149 bp. The crowdsourced results were highly concordant with 37 out of the 61 curators having at least 78% concordance with a set of expert curators, where there was 93% concordance amongst expert curators. This produced high confidence labels for 935 events. When compared to the heuristic-based draft benchmark SV callset from GIAB, the SVCurator crowdsourced labels were 94.5% concordant with the benchmark set. We found that curators can successfully evaluate putative SVs when given evidence from multiple sequencing technologies.

genomics↗

bioRxiv2018

Detection of GBA missense mutations and other variants using the Oxford Nanopore MinION

PurposeMutations in GBA cause Gaucher disease when biallelic, and are strong risk factors for Parkinsons disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA on the Oxford Nanopore MinION.\n\nMethodsWe sequenced an 8.9 kb amplicon from DNA samples of 17 individuals, including patients with Parkinsons and Gaucher disease, on older and current (R9.4) flow cells. These included samples with known mutations, assessed in a blinded fashion on the R9.4 data. We used NanoOK for quality metrics, two different aligners (Graphmap and NGMLR), Nanopolish and Sniffles to call variants, and Whatshap for phasing.\n\nResultsWe detected all known mutations, including the common p.N409S (N370S) and p.L483P (L444P), and three rarer ones, at the correct zygosity, as well as intronic SNPs. In a sample with the complex RecNciI allele, we detected an additional coding mutation, and a 55-base pair deletion. We confirmed compound heterozygosity where relevant. False positives were easily identified.\n\nConclusionThe Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantage of phasing and intronic analysis. It can be used as an efficient diagnostic tool.

genetics↗

bioRxiv2017

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array \"waves\", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

genomics↗