DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing.
BACKGROUND
DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number.


RESULTS
We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual.


CONCLUSIONS
The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.
Castle
John C
JC
Rosetta Inpharmatics LLC, Merck & Co., Inc., Seattle, Washington 98109, USA. castle@uni-mainz.de
Biery
Matthew
M
Bouzek
Heather
H
Xie
Tao
T
Chen
Ronghua
R
Misura
Kira
K
Jackson
Stuart
S
Armour
Christopher D
CD
Johnson
Jason M
JM
Rohl
Carol A
CA
Raymond
Christopher K
CK
D000818:Animals; D002276:Carcinoid Tumor / Q000235:genetics*; D045744:Cell Line, Tumor; D056915:DNA Copy Number Variations; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D016615:Telomere
Bioinformatics. 2009 Jul 15;25(14):1754-60
19451168
Nat Genet. 2008 Jun;40(6):722-9
18438408
Nat Genet. 2008 Oct;40(10):1199-203
18776910
Curr Opin Oncol. 2005 Mar;17(2):118-22
15725915
Nat Protoc. 2007;2(8):1973-86
17703209
Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):16156-61
12461184
Nat Methods. 2008 Jul;5(7):621-8
18516045
Nature. 2008 Nov 6;456(7218):66-72
18987736
Nat Genet. 2009 Oct;41(10):1061-7
19718026
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3089-94
14970324
Biochim Biophys Acta. 1998 Oct 14;1378(2):F115-77
9823374
Nat Methods. 2009 Jan;6(1):99-103
19043412
Cell. 1982 Aug;30(1):3-4
6751559
Proc Natl Acad Sci U S A. 2008 Oct 21;105(42):16266-71
18838674
J Biol Chem. 1974 Dec 25;249(24):7991-5
4473454
Genome Res. 2009 Sep;19(9):1586-92
19657104
Nature. 1990 Aug 30;346(6287):866-8
2392154
Biostatistics. 2010 Jan;11(1):164-75
19837654
Nature. 2008 Nov 6;456(7218):60-5
18987735
Nat Biotechnol. 2009 Feb;27(2):182-9
19182786