DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation
DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation
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.
Maserati Emanuela、Grace Colin、Valli Roberto、Nacheva Elizabeth、Taanman Jan-Willem、Proukakis Christos、Soenmez Aynur、Vattathil Selina、Mokretar Katya、Houlden Henry、Pittman Alan M、Schapira Anthony H、Ejaz Ayesha
Dipartimento di Medicina e Chirurgia, Universit¨¤dell?ˉInsubriaAcademic Haematology, Royal Free Campus, University College LondonDipartimento di Medicina e Chirurgia, Universit¨¤dell?ˉInsubriaAcademic Haematology, Royal Free Campus, University College LondonClinical Neuroscience, Institute of Neurology, University College LondonClinical Neuroscience, Institute of Neurology, University College LondonClinical Neuroscience, Institute of Neurology, University College LondonDepartment of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of AmericaAcademic Haematology, Royal Free Campus, University College London||Clinical Neuroscience, Institute of Neurology, University College LondonMolecular Neuroscience, Institute of Neurology, University College LondonMolecular Neuroscience, Institute of Neurology, University College LondonClinical Neuroscience, Institute of Neurology, University College LondonClinical Neuroscience, Institute of Neurology, University College London
基础医学遗传学分子生物学
Maserati Emanuela,Grace Colin,Valli Roberto,Nacheva Elizabeth,Taanman Jan-Willem,Proukakis Christos,Soenmez Aynur,Vattathil Selina,Mokretar Katya,Houlden Henry,Pittman Alan M,Schapira Anthony H,Ejaz Ayesha.DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation[EB/OL].(2025-03-28)[2025-08-02].https://www.biorxiv.org/content/10.1101/151126.点此复制
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