Biology subjects

Proukakis, C.

Publications and source records attributed to Proukakis, C..

2 recordsLinked to original sources

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↗