RT Journal Article
SR Electronic
T1 DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 151126
DO 10.1101/151126
A1 Elizabeth Nacheva
A1 Katya Mokretar
A1 Aynur Soenmez
A1 Alan M Pittman
A1 Colin Grace
A1 Roberto Valli
A1 Ayesha Ejaz
A1 Selina Vattathil
A1 Emanuela Maserati
A1 Henry Houlden
A1 Jan-Willem Taanman
A1 Anthony H Schapira
A1 Christos Proukakis
YR 2017
UL http://biorxiv.org/content/early/2017/06/16/151126.abstract
AB 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.