DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.
Elizabeth Nacheva,Katya Mokretar,Aynur Soenmez,Alan Pittman,Colin Grace,Roberto Valli,Ayesha Ejaz,Selina Vattathil,Emanuela Maserati,Henry Houlden,Jan-Willem Taanman,Anthony H.V. Schapira,Christos Proukakis +12 more
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TLDR
Evidence is provided for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues, and which may contribute to array “waves”, and could affect copy number determination, and sequencing coverage.Abstract:
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.read more
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Thinking outside the nucleus: Mitochondrial DNA copy number in health and disease
TL;DR: The existing literature which supports roles for inflammatory dynamics, immune function and alterations to cell signaling as consequences of variation in mtDNA-CN are reviewed, and it is proposed that future studies should focus on characterizing longitudinal, cell-type and cross-tissue profiles of mt DNA-CN.
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Evaluation of mitochondrial DNA copy number estimation techniques.
Ryan J. Longchamps,Christina A. Castellani,Stephanie Y. Yang,Charles E. Newcomb,Jason A Sumpter,John A. Lane,Megan L. Grove,Eliseo Guallar,Nathan Pankratz,Kent D. Taylor,Jerome I. Rotter,Eric Boerwinkle,Eric Boerwinkle,Dan E. Arking +13 more
TL;DR: The field moves towards more accurate methods for mtDNA-CN, as well as re-analyze trait associations as more WGS data becomes available from larger initiatives such as TOPMed.
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Review: Somatic mutations in neurodegeneration.
TL;DR: A model whereby the time of origin and spatial distribution of relevant somatic mutations, combined with any additional risk factors, may partly determine the development and onset age of sporadic neurodegenerative diseases is presented.
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Somatic copy number gains of α-synuclein (SNCA) in Parkinson's disease and multiple system atrophy brains.
Katya Mokretar,Katya Mokretar,Daniel Pease,Jan-Willem Taanman,Aynur Soenmez,Ayesha Ejaz,Tammaryn Lashley,Helen Ling,Steve M. Gentleman,Henry Houlden,Janice L. Holton,Anthony H.V. Schapira,Elizabeth Nacheva,Christos Proukakis +13 more
TL;DR: Evidence of somatic SNCA gains in brain, more commonly in nigral dopaminergic neurons of Parkinson's disease than controls, negatively correlated with onset age, and possibly commonest in some multiple system atrophy cases is presented.
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Evaluation of the detection of GBA missense mutations and other variants using the Oxford Nanopore MinION.
Melissa Leija-Salazar,Fritz J. Sedlazeck,Marco Toffoli,Stephen Mullin,Stephen Mullin,Katya Mokretar,Maria Athanasopoulou,Aimee Donald,Reena Sharma,Derralynn Hughes,Anthony H.V. Schapira,Christos Proukakis +11 more
TL;DR: This work designs and validate a method for sequencing GBA using long reads and proves its ability to identify mutations in Gaucher disease when biallelic and Parkinson's disease when heterozygous.
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