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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 M Pittman, Colin Grace, Roberto Valli, Ayesha Ejaz, Selina Vattathil, Emanuela Maserati, Henry Houlden, Jan-Willem Taanman, Anthony H Schapira, View ORCID ProfileChristos Proukakis
doi: https://doi.org/10.1101/151126
Elizabeth Nacheva
1Academic Haematology, Royal Free Campus, University College London, London, UK.
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Katya Mokretar
1Academic Haematology, Royal Free Campus, University College London, London, UK.
2Clinical Neuroscience, Institute of Neurology, University College London, London, UK.
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Aynur Soenmez
2Clinical Neuroscience, Institute of Neurology, University College London, London, UK.
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Alan M Pittman
3Molecular Neuroscience, Institute of Neurology, University College London, London, UK.
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Colin Grace
1Academic Haematology, Royal Free Campus, University College London, London, UK.
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Roberto Valli
4Dipartimento di Medicina e Chirurgia, Universitàdell’Insubria, Varese, Italy.
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Ayesha Ejaz
2Clinical Neuroscience, Institute of Neurology, University College London, London, UK.
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Selina Vattathil
5Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
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Emanuela Maserati
4Dipartimento di Medicina e Chirurgia, Universitàdell’Insubria, Varese, Italy.
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Henry Houlden
3Molecular Neuroscience, Institute of Neurology, University College London, London, UK.
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Jan-Willem Taanman
2Clinical Neuroscience, Institute of Neurology, University College London, London, UK.
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Anthony H Schapira
2Clinical Neuroscience, Institute of Neurology, University College London, London, UK.
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Christos Proukakis
2Clinical Neuroscience, Institute of Neurology, University College London, London, UK.
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  • ORCID record for Christos Proukakis
  • For correspondence: c.proukakis@ucl.ac.uk
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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.

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Posted June 16, 2017.
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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 M Pittman, Colin Grace, Roberto Valli, Ayesha Ejaz, Selina Vattathil, Emanuela Maserati, Henry Houlden, Jan-Willem Taanman, Anthony H Schapira, Christos Proukakis
bioRxiv 151126; doi: https://doi.org/10.1101/151126
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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 M Pittman, Colin Grace, Roberto Valli, Ayesha Ejaz, Selina Vattathil, Emanuela Maserati, Henry Houlden, Jan-Willem Taanman, Anthony H Schapira, Christos Proukakis
bioRxiv 151126; doi: https://doi.org/10.1101/151126

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