BACKGROUND
dna copy number variations occur within populations and aberrations can cause tumors, be used for drug target identification, and be used as biomarkers of tumor drug response.
egfr  amplification, for instance, is a marker for gefitinib treatment  <cit>  and tyms  amplification conveys 5-fluorouracil resistance in colon tumors  <cit> .
the next-generation sequence enables generation of millions of short sequence tags in a single experiment.
using dna as an input, the technology has been used to resequence entire genomes, including from normal individuals  <cit>  and from cancerous cells  <cit> , and to resequence targeted genomic regions, such as resequencing protein coding regions to discover somatic mutations  <cit> .
alternatively, using rna as an input, the technology has been used to profile rna expression levels, where the number of sequence reads "tagging" an rna transcript is a measure of its expression  <cit> .
combining these ideas and building on previous methods  <cit> , this report describes the development of a sequencing-based platform to profile "expression" of dna, inputting dna and analyzing the resultant data using algorithms previously established for gene expression profiling.
the platform resolution and cost are tunable; is lab automatable; can be used for both discovery of novel and profiling of known dna copy number variations; outputs copy number and uncertainty as opposed to ratios; and can be used to profile mitochondria and telomeric dna.
RESULTS
application 1: assaying nuclear genomic copy number
we assayed the genomic copy number in a pool of dna derived from blood from non-diseased males; a pool of dna derived from blood from non-diseased females; and dna from umc- <dig> cells, a lung carcinoid-derived cell line.
we generated and sequenced each library, aligned resultant reads to the genome, and selected reads aligning to only one genomic location .
across chromosome  <dig>  the number of reads from the male pool mapping to each genomic block is near  <dig> .
from the female pool, the number of reads mapping to each block is similarly constant, at roughly  <dig> .
we tested normalization by the gc content in each dna block  <cit> ; however, we found that the relative number of reads mapping to a block depended more strongly on molecular biology protocols.
instead, we developed and applied a novel normalization method using the male pool as a reference, and with this normalization were able to derive the copy number, upper and lower bounds, and the significance of any deviation .
in the female dna pool, the copy number is two across the entire chromosome, with a single exception at position ~ <dig> mb .
use of a single reference sample, processed using the same bench protocol as used for the non-reference samples, therefore eliminates the need to normalize for gc content and for sequence uniqueness within each block.
conversely, umc- <dig> cells show dramatic copy number variations.
before normalization, the number of reads mapping to each block shows many discrete levels, including blocks with no reads and blocks with roughly  <dig>   <dig>  and  <dig> reads .
after normalization, these discrete levels map to copy number of zero, one, two, three, and four .
intriguingly, the homozygously deleted region spanning from  <dig> to  <dig> mb includes the putative tumor suppressor cdkn2a  <cit>  .
across chromosome  <dig>  the female pool has copy number two across the entire chromosome, as expected .
the umc- <dig> cell line is highly aberrant: copy number starts at three; is amplified to 9; falls to three; is amplified up to  <dig> copies; drops to three; falls to two; has a small segment at three copies; back to two; and then back to three.
again intriguingly, the highly amplified region includes the tumor-associated gene kras  <cit> .
this assay provides a genome-wide view of copy number in a single assay, allowing the full extent of copy number within a sample to be assessed .
while chromosomes  <dig>   <dig>   <dig>   <dig>  and x in umc- <dig> cells show little disruption, many chromosomes show amazing copy number variation.
almost  <dig> mb of chromosome  <dig> is present at  <dig> copies.
an  <dig> mb block of chromosome  <dig> is present at one copy.
many changes in copy number occur close to or across centromeres.
we also identify that the cell line is derived from a male patient as chromosome x is present at one copy and many reads map to chromosome y.
to validate findings, we assayed copy number with qpcr at  <dig> locations .
the five locations predicted to have copy number two by sequencing indeed had copy number two as measured by qpcr.
copy number measurements of  <dig>   <dig>   <dig>  and  <dig> also validated.
at high copy number, sequencing and qpcr measurements were similar:  <dig> and 11;  <dig> and 24;  <dig> and  <dig>  respectively.
these results also show high correspondence with findings from an array-based platform  <cit>  , including the chromosome  <dig> deletion near  <dig> mb and the small amplification near position  <dig> mb.
results from the highly amplified region of chromosome  <dig> qualitatively agree; however, the array-based method predicts only  <dig> copy number in the region where the sequencing and qpcr assays measure over  <dig> copy number .
across the entire genome, the array-based data for umc- <dig> cells, downloaded from the sanger center and analyzed with the picnic algorithm  <cit> , show high concordance at copy number five and below, but discrepancies at higher copy number .
the array-platform returns lower copy numbers than found by qpcr and sequencing, potentially the result of microarray probe saturation  <cit> .
values are rounded to the nearest integer.
application 2: assaying genetic loci copy number
conceptually, one can define biological elements in terms of gene loci rather than genomic blocks, and thus, by counting the number of reads uniquely mapping to each locus, generate a gene-copy number table.
here, we defined locus coordinates as the greater of the transcript start-to-stop span or  <dig> kb centered on the loci.
as before, we normalized the counts to the reference male pool, assuming the male pool is diploid across autosomes and haploid across allosomes.
the loci with the most significant p-values for higher copy number in the female pool are, not surprisingly, found on chromosome x .
in each case, this platform assayed the loci at copy number two in the female pool and one in the male pool and umc- <dig> cells.
the loci with the most significant lower copy number in umc- <dig> cells are in the chromosome  <dig> deletion , where the loci show copy number two in the female pool.
the loci with the most significant amplifications are in the chromosome  <dig> amplification , with copy number at almost  <dig>
similarly, biological elements can be defined in terms of the coordinates of known dna copy number polymorphisms  <cit> .
by counting reads aligning within the coordinates of each established polymorphism, one can monitor population-variable copy number polymorphisms .
application 3: mitochondrial copy number
biological elements can also be defined as the mitochondrial dna , in which case one assays the number of mitochondria in the cells.
while the counts of mtdna within each individual cell will vary, blood-derived cells on average contain fewer mitochondria relative to cell lines  <cit> .
from the aligned sequence reads, we counted and normalized the number of reads aligning to mtdna as a measure of the average mtdna levels within each sample.
indeed, we found that the umc- <dig> cells contain over  <dig> times more mitochondria than the blood-derived male and female pools .
application 4: telomere copy number
finally, another fascinating biological element that can be interrogated is telomeric sequence.
telomeres protect the ends of chromosomes; are on average shorter in cells that have undergone many divisions, such as older cells, tumors, and cell lines; and comprise repetitive ttaggg motifs  <cit> .
we counted and normalized the number of sequence reads containing  <dig>  strikingly, the umc- <dig> cells do contain significantly fewer telomere-associated reads than either the female or male pools .
discussion and
CONCLUSIONS
assaying dna copy number by next-generation sequencing is robust and accurate.
the method described here requires a simple genomic dna library construction; returns integer copy number values for homogeneous cells; and has a large dynamic range.
the platform is unbiased in the sense that genomic targets are not preselected, such as is the case with qpcr and microarrays, and thus, given a new genome assembly, a new set of copy number polymorphisms, or a new set of biological dna elements, the sequence reads maintain utility through re-alignment.
our research builds on previous efforts.
the 'digital karyotyping' protocol uses restriction enzymes and sage sequencing technology to generate reads that have been used to measure copy number variation and detect infectious viral dna <cit> .
using illumina deep-sequencing, fetal aneuploidy was assayed, identifying down, edward, and patau syndromes based on chromosome-specific trisomy  <cit> .
illumina deep-sequencing has been used to examine copy number across nuclear chromosomes.
campbell et al <cit>  used paired-end reads from size-selected libraries to identify genomic structural rearrangements and, integrating estimates of copy number with paired-end reads mapping to distal locations, were able to identify breakpoint coordinates and novel dna sutures.
recently, chiang et al <cit>  sequenced a size-selected library and measured the log-ratio change between normal and tumor sample pairs across nuclear chromosomes.
they elegantly show trade-off curves between read number, copy number change, and genomic resolution and show statistical determination of breakpoints.
in comparing their results to array-based methods, they find the sequencing-based platform has a larger dynamic range.
yoon et al.
<cit>  and alkan et al.
<cit>  developed similar methods for use in human resequencing projects, using over  <dig> billion reads and 30× genome coverage to identify copy number polymorphisms in disease-free cells.
the method described here expands on these groundbreaking studies in several ways.
first, our library construction does not include a size selection and is thus lab automatable.
second, by using a single diploid reference sample along with a novel normalization algorithm, our method removes biases inherent in molecular biology protocols and outputs absolute copy number in addition to log-ratio values.
third, we defined an uncertainty that allows us to estimate upper and lower bounds and p-values for each copy number measurement, both for absolute and relative measurements.
forth, by defining biological elements as not only nuclear dna blocks positioned evenly across the nuclear genome, this platform enables assaying other biologically meaningful dna elements, including gene loci, known copy number polymorphisms, mitochondria, and telomeres.
finally, these results were generated using an illumina genome analyzer ii instrument in june,  <dig>  with one sample per lane, resulting in only  <dig> to  <dig> million  <dig> nt reads per sample.
sequencing instrumentation continues to improve, allowing more reads at lower costs.
as the resolution of this assay is inversely proportional to the number of aligned reads, sample multiplexing and increasing numbers of sequence reads will enable increased resolution and/or significantly decreased costs.

 dna copy number variations occur within populations and aberrations can cause tumors, be used for drug target identification, and be used as biomarkers of tumor drug response. egfr amplification, for instance, is a marker for gefitinib treatment and tyms amplification conveys 5-fluorouracil resistance in colon tumors. the next-generation sequence enables generation of millions of short sequence tags in a single experiment. using dna as an input, the technology has been used to resequence entire genomes, including from normal individuals and from cancerous cells, and to resequence targeted genomic regions, such as resequencing protein coding regions to discover somatic mutations. alternatively, using rna as an input, the technology has been used to profile rna expression levels, where the number of sequence reads "tagging" an rna transcript is a measure of its expression. combining these ideas and building on previous methods, this report describes the development of a sequencing-based platform to profile "expression" of dna, inputting dna and analyzing the resultant data using algorithms previously established for gene expression profiling. the platform resolution and cost are tunable; is lab automatable; can be used for both discovery of novel and profiling of known dna copy number variations; outputs copy number and uncertainty as opposed to ratios; and can be used to profile mitochondria and telomeric dna.  application 1: assaying nuclear genomic copy number we assayed the genomic copy number in a pool of dna derived from blood from non-diseased males; a pool of dna derived from blood from non-diseased females; and dna from umc- cells, a lung carcinoid-derived cell line. we generated and sequenced each library, aligned resultant reads to the genome, and selected reads aligning to only one genomic location. across chromosome the number of reads from the male pool mapping to each genomic block is near. from the female pool, the number of reads mapping to each block is similarly constant, at roughly. we tested normalization by the gc content in each dna block; however, we found that the relative number of reads mapping to a block depended more strongly on molecular biology protocols. instead, we developed and applied a novel normalization method using the male pool as a reference, and with this normalization were able to derive the copy number, upper and lower bounds, and the significance of any deviation. in the female dna pool, the copy number is two across the entire chromosome, with a single exception at position ~ mb. use of a single reference sample, processed using the same bench protocol as used for the non-reference samples, therefore eliminates the need to normalize for gc content and for sequence uniqueness within each block. conversely, umc- cells show dramatic copy number variations. before normalization, the number of reads mapping to each block shows many discrete levels, including blocks with no reads and blocks with roughly and reads. after normalization, these discrete levels map to copy number of zero, one, two, three, and four. intriguingly, the homozygously deleted region spanning from to mb includes the putative tumor suppressor cdkn2a. across chromosome the female pool has copy number two across the entire chromosome, as expected. the umc- cell line is highly aberrant: copy number starts at three; is amplified to 9; falls to three; is amplified up to copies; drops to three; falls to two; has a small segment at three copies; back to two; and then back to three. again intriguingly, the highly amplified region includes the tumor-associated gene kras. this assay provides a genome-wide view of copy number in a single assay, allowing the full extent of copy number within a sample to be assessed. while chromosomes and x in umc- cells show little disruption, many chromosomes show amazing copy number variation. almost mb of chromosome is present at copies. an mb block of chromosome is present at one copy. many changes in copy number occur close to or across centromeres. we also identify that the cell line is derived from a male patient as chromosome x is present at one copy and many reads map to chromosome y. to validate findings, we assayed copy number with qpcr at locations. the five locations predicted to have copy number two by sequencing indeed had copy number two as measured by qpcr. copy number measurements of and also validated. at high copy number, sequencing and qpcr measurements were similar: and 11; and 24; and respectively. these results also show high correspondence with findings from an array-based platform, including the chromosome deletion near mb and the small amplification near position mb. results from the highly amplified region of chromosome qualitatively agree; however, the array-based method predicts only copy number in the region where the sequencing and qpcr assays measure over copy number. across the entire genome, the array-based data for umc- cells, downloaded from the sanger center and analyzed with the picnic algorithm, show high concordance at copy number five and below, but discrepancies at higher copy number. the array-platform returns lower copy numbers than found by qpcr and sequencing, potentially the result of microarray probe saturation. values are rounded to the nearest integer. application 2: assaying genetic loci copy number conceptually, one can define biological elements in terms of gene loci rather than genomic blocks, and thus, by counting the number of reads uniquely mapping to each locus, generate a gene-copy number table. here, we defined locus coordinates as the greater of the transcript start-to-stop span or kb centered on the loci. as before, we normalized the counts to the reference male pool, assuming the male pool is diploid across autosomes and haploid across allosomes. the loci with the most significant p-values for higher copy number in the female pool are, not surprisingly, found on chromosome x. in each case, this platform assayed the loci at copy number two in the female pool and one in the male pool and umc- cells. the loci with the most significant lower copy number in umc- cells are in the chromosome deletion, where the loci show copy number two in the female pool. the loci with the most significant amplifications are in the chromosome amplification, with copy number at almost similarly, biological elements can be defined in terms of the coordinates of known dna copy number polymorphisms. by counting reads aligning within the coordinates of each established polymorphism, one can monitor population-variable copy number polymorphisms. application 3: mitochondrial copy number biological elements can also be defined as the mitochondrial dna, in which case one assays the number of mitochondria in the cells. while the counts of mtdna within each individual cell will vary, blood-derived cells on average contain fewer mitochondria relative to cell lines. from the aligned sequence reads, we counted and normalized the number of reads aligning to mtdna as a measure of the average mtdna levels within each sample. indeed, we found that the umc- cells contain over times more mitochondria than the blood-derived male and female pools. application 4: telomere copy number finally, another fascinating biological element that can be interrogated is telomeric sequence. telomeres protect the ends of chromosomes; are on average shorter in cells that have undergone many divisions, such as older cells, tumors, and cell lines; and comprise repetitive ttaggg motifs. we counted and normalized the number of sequence reads containing strikingly, the umc- cells do contain significantly fewer telomere-associated reads than either the female or male pools. discussion and  assaying dna copy number by next-generation sequencing is robust and accurate. the method described here requires a simple genomic dna library construction; returns integer copy number values for homogeneous cells; and has a large dynamic range. the platform is unbiased in the sense that genomic targets are not preselected, such as is the case with qpcr and microarrays, and thus, given a new genome assembly, a new set of copy number polymorphisms, or a new set of biological dna elements, the sequence reads maintain utility through re-alignment. our research builds on previous efforts. the'digital karyotyping' protocol uses restriction enzymes and sage sequencing technology to generate reads that have been used to measure copy number variation and detect infectious viral dna. using illumina deep-sequencing, fetal aneuploidy was assayed, identifying down, edward, and patau syndromes based on chromosome-specific trisomy. illumina deep-sequencing has been used to examine copy number across nuclear chromosomes. campbell et al used paired-end reads from size-selected libraries to identify genomic structural rearrangements and, integrating estimates of copy number with paired-end reads mapping to distal locations, were able to identify breakpoint coordinates and novel dna sutures. recently, chiang et al sequenced a size-selected library and measured the log-ratio change between normal and tumor sample pairs across nuclear chromosomes. they elegantly show trade-off curves between read number, copy number change, and genomic resolution and show statistical determination of breakpoints. in comparing their results to array-based methods, they find the sequencing-based platform has a larger dynamic range. yoon et al. and alkan et al. developed similar methods for use in human resequencing projects, using over billion reads and 30× genome coverage to identify copy number polymorphisms in disease-free cells. the method described here expands on these groundbreaking studies in several ways. first, our library construction does not include a size selection and is thus lab automatable. second, by using a single diploid reference sample along with a novel normalization algorithm, our method removes biases inherent in molecular biology protocols and outputs absolute copy number in addition to log-ratio values. third, we defined an uncertainty that allows us to estimate upper and lower bounds and p-values for each copy number measurement, both for absolute and relative measurements. forth, by defining biological elements as not only nuclear dna blocks positioned evenly across the nuclear genome, this platform enables assaying other biologically meaningful dna elements, including gene loci, known copy number polymorphisms, mitochondria, and telomeres. finally, these results were generated using an illumina genome analyzer ii instrument in june, with one sample per lane, resulting in only to million nt reads per sample. sequencing instrumentation continues to improve, allowing more reads at lower costs. as the resolution of this assay is inversely proportional to the number of aligned reads, sample multiplexing and increasing numbers of sequence reads will enable increased resolution and/or significantly decreased costs. 
the described assay outputs absolute copy number, outputs an error estimate, and is more accurate than array-based platforms at high copy number. to demonstrate this platform, we assayed umc- cells using million nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to copies; times more mitochondria and times less telomeric sequence than a pool of non-diseased, blood-derived dna; and that umc- was derived from a male individual. the assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads. dna copy number variations occur within populations and aberrations can cause disease.
 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.  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- cells using million nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to copies; times more mitochondria and times less telomeric sequence than a pool of non-diseased, blood-derived dna; and that umc- was derived from a male individual.  the described assay outputs absolute copy number, outputs an error estimate, 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. 