A mechanistic basis for amplification differences between samples and between genome regions
© Veal et al.; licensee BioMed Central Ltd. 2012
Received: 1 May 2012
Accepted: 28 August 2012
Published: 5 September 2012
For many analytical methods the efficiency of DNA amplification varies across the genome and between samples. The most affected genome regions tend to correlate with high C + G content, however this relationship is complex and does not explain why the direction and magnitude of effects varies considerably between samples.
Here, we provide evidence that sequence elements that are particularly high in C + G content can remain annealed even when aggressive melting conditions are applied. In turn, this behavior creates broader ‘Thermodynamically Ultra-Fastened’ (TUF) regions characterized by incomplete denaturation of the two DNA strands, so reducing amplification efficiency throughout these domains.
This model provides a mechanistic explanation for why some genome regions are particularly difficult to amplify and assay in many procedures, and importantly it also explains inter-sample variability of this behavior. That is, DNA samples of varying quality will carry more or fewer nicks and breaks, and hence their intact TUF regions will have different lengths and so be differentially affected by this amplification suppression mechanism – with ‘higher’ quality DNAs being the most vulnerable. A major practical consequence of this is that inter-region and inter-sample variability can be largely overcome by employing routine fragmentation methods (e.g. sonication or restriction enzyme digestion) prior to sample amplification.
KeywordsDNA amplification DNA denaturation C + G Illumina infinium
The fact that amplification methods vary in efficiency across the genome has often been noted, for example in whole genome amplification (WGA), next generation sequencing, genome wide SNP genotyping, and PCR [1–5]. Difficult to assay regions are somewhat correlated with high C + G content [1, 6–10], but this relationship is complex, DNA sample dependent, and incompletely understood. Regions of high C + G content tend to resist the essential DNA denaturation step at the initiation of nearly all DNA amplification protocols, though it is assumed that this effect will not be so extreme as to completely prevent DNA strand separation. However, this assumption may be incorrect. In DNA melting studies in the early 1970s, select human genome DNA fragments were seen to remain double stranded under extreme denaturing conditions [11, 12]. The nature of these challenging sequences has not yet been determined, and today most investigators are probably unaware of the early reports.
Here, we investigate a number of genomic regions that across several samples produce low intensity hybridization in Illumina Infinium genotyping. We find that a major factor that can influence such regions are intervals of high C + G content that do not denature efficiently under routinely used conditions. These intervals cause connected DNA sequences to rapidly re-anneal and prevent access to primers or probes. The effects of this in PCR could be completely ameliorated by enzymatic separation of the high C + G interval and the assay target. We postulate that inter-sample variability is due to the amount and random distribution of nicking within a DNA sample which acts to separate these difficult to denature sequences from other DNA, and that highly intact DNAs will suffer the most. We provide optimized PCR protocols and suggest that DNA is pretreated by either sonication or restriction enzyme digestion prior to amplification steps in methods.
Results and discussion
Testing DNA melting using southern blot hybridisation
Primers and co-ordinates for all PCR amplicons and Probes (hg18, GRCh36)
Forward primer sequence
Reverse primer sequence
Reduced PCR amplification efficiency assessed by PRT
We examined normal and ‘weak Illumina signal’ regions using the Paralogue Ratio Test (PRT) [13, 14]. Standard PRT, which is a powerful technique to genotype copy number variation, employs a single pair of PCR primers to co-amplify a ‘test’ locus (whose copy number is being assessed) and a ‘reference’ locus (a stable single copy sequence) in a single PCR reaction. The two amplicons are distinguished by size, and their relative product amounts used to determine the test locus copy number. We adapted this concept to co-amplify single copy sequences from normal and ‘weak Illumina signal’ regions. This allowed the comparison of their relative amplification efficiencies in the same PCR reaction with identical conditions and DNA template concentration. Importantly, the ‘test’ and ‘reference’ amplicons employed for six assay designs created for these experiments had similar and not unusually high C + G content (average values of 56.8 and 51.0% C + G respectively). In all six assays, the ‘reference’ amplicon (i.e., the product amplified from the assay’s normal Illumina signal region) produced a strong band, whereas its partnered ‘test’ amplicon produced a weaker band (typically 10-50% of the strength of the reference), indicating a reduced PCR efficiency for ‘weak Illumina signal’ regions.
Enhancing denaturing conditions improves amplification
Regions of high C + G serve as nuclei for rapid re-annealing of neighboring DNA sequences
Genome wide patterns of TUF
Association between PRT performance and Illumina Infinium intensity correlations with C + G and CpG for 54 samples
C + G
These observations imply that it should be possible to bioinformatically predict and partially correct for the effects of TUF areas of the genome and for other phenomena that have been observed to induce similar C + G correlated effects. Diskin et al.  demonstrate that C + G-correlated intensity fluctuations (waves) are present in both Illumina and Affymetrix whole-genome SNP microarrays and that C + G content in 1 Mb windows are highly correlated with intensity (both positively and negatively) with the amplitude determined by the degree that DNA quantity/concentration deviated from the vendor’s recommended level. Efficiency of PCR amplification of short DNA fragments (<200 bp) has also been shown to be affected by local C + G-content and some suggestions have been made on how to predict and compensate for such effects .
Artificial generation or repair of DNA nicking/fragmentation
In summary, our description of TUF represents the important recognition of a phenomenon relevant to many regions of the genome, thus impacting in a sample dependant manner the conduct of genome-wide studies of distinct types of genetic variation in relation to human diseases/traits. For example, it may well be practically relevant in Copy Number Variation (CNV) research and the use of next generation sequencing, where assay behavior can be unpredictable [25–28]. Further work will be required to fully understand the biochemical basis of the TUF regions in order to optimally develop protocols and approaches for large scale genomic analyses. Knowledge of the TUF phenomenon and ways to overcome its deleterious consequences should provide investigators with a more nuanced approach towards handling issues related to C + G content and its effect upon assay robustness and efficiency.
Human genomic DNA samples
DNA donors for Southern Blotting and PRT analysis of TUF regions were of north European origin, and had given informed consent with ethical approval from the Leicestershire, Northamptonshire and Rutland Research Ethics Committee (LNRREC Ref. No. 6659 UHL). DNA was prepared from fresh blood as follows. 20 ml whole blood was centrifuged at 1300 g at 4°C for 15 minutes. The buffy coat was extracted and incubated at 37°C in 15 ml lysis buffer (10 mM Tris-Cl (pH 8.0) 0.1 M EDTA (pH 8.0) 0.5% w/v SDS) for 1 hour. Proteinase K (final concentration 100 μg/ml) was added and mixed gently followed by incubation at 50°C overnight. After allowing to cool to room temperature an equal volume of phenol equilibrated with 0.1 M Tris HCl and mixed slowly on a Stuart Rotator SB3 for 10 mins. The phases were separated by centrifugation at 5600 g for 15 min. The aqueous phase was transferred to a fresh tube and the phenol extraction repeated twice. To the final aqueous phase 1/10th volume 5 M Ammonium Acetate and 2 volumes of 100% Ethanol were added. Samples were mixed very slowly and carefully by inversion. The precipitated DNA was spooled using a glass hook and dried briefly and dissolved in water to a final concentration of 200 ng/μl. DNA quality and quantity was assessed by gel electrophoresis and on the NanoDrop ND-8000 spectrophotometer.
Paralogue ratio test (PRT)
PRTs were designed according to information from Armour et al.,. All PRT oligonucleotide primers are described in Table 1. 10 μl PRT PCRs contained 1 x PCR buffer (75 mM Tris HCl (pH8.8), 20 mM (NH4)2SO4, 0.01% v/v Tween) (Abgene, Epsom, Surrey, UK), 1.5 mM MgCl2 (Abgene), 0.15 μM of each primer (Biomers), 0.2 mM dNTPs (Promega), 0.3 U Taq polymerase (Kapa Biosystems, Boston, MA, USA) and 10 to 25 ng DNA. PCR were initially heated to 94°C for 30 seconds, and then heated for 25 to 35 cycles as follows: 94°C for 30 seconds; annealing temperature for 30 seconds; 72°C for 1 minute. A final extension was carried out at 72°C for 5 minutes. Where required, restriction enzyme digests were performed to allow visualisation of similar sized PRT products. On using additives (DMSO up to 50%, betaine up to 2 M) the optimal annealing temperature was re-optimised for each assay. Recommended PCR conditions for TUF regions are 1.5 M betaine, 5U/μl Taq polymerase, 0.01U/μl pfu enzyme and use of 98°C denaturing temperature in all cycles. Higher concentrations of betaine may be appropriate for individual PCRs.
Agarose gel peak height quantification
Gels were documented using a GBOX HR, Gel documentation system (Syngene, Cambridge, Cambridgeshire, UK) using the EDR function and the maximum resolution settings (5.52 M pixels). Peaks were identified and peak heights quantified using the Gene Tools programme version 4.00 (A) (Syngene). For peak height analysis, the rolling disc method (diameter = 30 pixels) was used to determine peak base line.
Pre-PCR heat denaturation
High temperature denaturing was performed in a 96 well format heat block set to the desired temperature. Sierra Antifreeze/coolant (Peak performance products, Northbrook, IL, USA) was used to maintain a liquid contact between the tubes, thermometer and heat block. The DNA was denatured in either water or in buffered conditions (1 x PCR buffer, as above) in tubes with the lids sealed tightly with Nescofilm to prevent evaporation at temperatures greater than 100°C. Samples were heated for 1 minute and snap cooled on ice for 5 minutes. Samples were stored at −20°C and thawed on ice prior to use.
Sonication of DNA
Aliquots of genomic DNA (200 ng/μl) were sonicated for 30 second intervals (with a 30 second gap), using a Bioruptor (Diagenode, Liège, Belgium) until the desired size range (0.3 to 3.0 kbp) was reached (visualised by agarose gel electrophoresis).
Adapted illumina protocol
Using conditions recommended by Illumina, 200 ng samples of genomic DNA (with or without pre-processing as necessary for each experiment) were hybridised to human370CNV Infinium HD BeadChips (Illumina INC, San Diego, CA, USA).
Whole genome amplification
Whole genome amplification was performed using the REPLI-g Mini Kit (Qiagen) to amplify a range of masses of human genomic DNA to generate >8 μg of DNA. Samples were prepared using the isothermal amplification reaction in PCR tubes incubated at 30°C for 16 hours and 65°C for 3 minutes in a thermal cycler. Amplified products were quantified using a NanoDrop spectrophotometer and visualised on a 0.8% LE agarose gel with Ethidium Bromide.
Restriction enzyme digestion for southern blotting
Six μg of genomic DNA was digested using selected enzymes supplied by New England Biolabs (NEB) (Hitchin, Hertfordshire, UK) under the conditions recommended by the supplier with the addition of 4 mM Spermidine pH 7.4. Double digests were performed in the most suitable buffer, and the quantity of the least active enzyme per reaction was doubled if required.
DNA denaturing prior to southern blotting
Heat denaturation was performed in a water-bath at 100°C for either for 40 seconds to 4 minutes as stated. Samples were snap cooled on ice for 5 minutes prior to gel electrophoresis.
Alkaline denaturation was performed by addition of 0.4 M NaOH to 0.32 M (~ 240 μl added to 54 μl of sample), and incubation at room temperature for 10 minutes. 1 M Tris Hcl (pH 8) was added to 0.02 M prior to neutralisation (pH 8 to 8.5) with 0.4 M HCl. Samples were ethanol precipitated and dissolved in distilled water.
Southern blotting and hybridisation
Digested DNA was run at 3 V/cm in 0.7% agarose gels (LE agarose, Seakem. 1 X TAE (4.84 g Tris base, 11.4 ml glacial acetic acid, 3.7 g EDTA pH 8.0 per litre)). The resulting gels were soaked twice in denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 30 minutes, and twice in neutralising solution (0.5 M Tris pH 7.2, 1 M NaCl) for 30 min. The denatured DNA was transferred onto uncharged nylon membranes (MAGNA, Nylon, Transfer Membrane, 0.45 Micron; GE Water & Process Technologies, Trevose, PA, USA) using 10X SSC as the transfer buffer and fixed to the membranes by baking at 80°C in a Sanyo MOV drying oven (Sanyo E&E Europe BV, Biomedical Division, Loughborough, Leicestershire, UK), for 1 hour.
PCR amplified probes (Table 1) were purified using a Qiagen MinElute PCR purification kit (Qiagen). 75 ng of probe was labelled for 15 minutes with α-32P –dCTP (Perkin Elmer, Waltham, MA USA) using the Rediprime II random prime labelling system (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK), purified using ILLUSTA NICK Columns Sephadex DNA grade (GE Healthcare, Little Chalford, Buckinghamshire, UK), and eluted in 400 μl column wash (1 x TE, 0.1% w/v SDS). 75 μg of human Cot I DNA (Invitrogen, Paisley, Renfrewshire, UK) was added prior to denaturation at 100°C for 6 minutes and snap cooling on ice for 5 minutes.
Hybridisation was performed in 20 ml Church buffer (0.5 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, 1% BSA ) with 2 mg heat denatured (100°C for 5 min, ice for 5 min) salmon sperm DNA. Pre-hybridisation was performed at 65°C in a rolling bottle for 2 hours prior to hybridisation for 10 hours. Hybridised blots were washed for 10 min at 65°C in 0.1 x SSC, 0.1% SDS. Counts were recorded using a phosphoimager screen (Amersham Biosciences) for between 12 and 60 hours. Further washing at 68°C or 72°C depending on the number of background counts.
Regression analysis of LRR and G + C/CpG content for varying window sizes
The log probe intensity ratio (LRR) value for each SNP or CNV assay provides data on probe intensity relative to that of the estimated genotype-specific cluster location. LRR values estimated by the Genome Studio software were corrected for bias due to the properties of the assay chemistry and fluorescent dyes used in the probes. We implemented a method similar to that described by Staaf et al.  to re-estimate LRR after applying quantile-normalization, with an enhanced multiple linear regression model, incorporating within-chip signal re-scaling terms and a polynomial correction for GC and CpG waves. The correction model is an extension to the method described in Diskin et al. with terms for multiple window sizes for proportion of GC and CpG content around the genomic location of each set of probes. GC and CpG terms in the regression model are the proportion of GC and CpG content for window sizes (in bp) of 50, 100, 500, 1 k, 10 k, 50 k, 100 k, 250 k, and 1 M centered around the genomic location of each assay, based on locations annotated in the Illumina manifest files and sequence context based on the NCBI build 36 reference genome sequence. This model is estimated per sample, as the phenomenon is modulated by TUF, the concentration of the DNA input, and possibly other factors. The final LRR was re-computed using the resulting quantile-normalized and GC/CpG corrected values as shown in Peiffer et al.. The reduction in variance of the LRR values is shown in Figure 6.
Whole genome amplification
Single nucleotide polymorphism
Polymerase chain reaction
Paralogue ratio test
Copy number variation.
This research was supported by Action Medical Research (grants SP4139 and SP4483) and by the European Union’s Seventh Framework Programme (FP7/2007-2013) project READNA (grant agreement HEALTH-F4-2008-201418). We wish to recognize earlier collaborations particularly with Nancy J Cox from the Division of Biological Sciences, University of Chicago and Paul H Dear and Bernard Konfortov of the MRC laboratory of Molecular Biology (University of Cambridge) that contributed to pointing our experiments towards identifying the TUF phenomenon. The authors wish to thank Ed Schwalbe (University of Newcastle) and Nathalie Zahra (University of Leicester) for their technical support.
- Pugh TJ, Delaney AD, Farnoud N, Flibotte S, Griffith M, Li HI, Qian H, Farinha P, Gascoyne RD, Marra MA: Impact of whole genome amplification on analysis of copy number variants. Nucleic Acids Res. 2008, 36: e80-10.1093/nar/gkn378.PubMed CentralView ArticlePubMedGoogle Scholar
- Dickson PA, Montgomery GW, Henders A, Campbell MJ, Martin NG, James MR: Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan. Nucleic Acids Res. 2005, 33: e119-10.1093/nar/gni126.PubMed CentralView ArticlePubMedGoogle Scholar
- Bergen AW, Qi Y, Haque KA, Welch RA, Chanock SJ: Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance. BMC Biotechnol. 2005, 5: 24-10.1186/1472-6750-5-24.PubMed CentralView ArticlePubMedGoogle Scholar
- Cunningham JM, Sellers TA, Schildkraut JM, Fredericksen ZS, Vierkant RA, Kelemen LE, Gadre M, Phelan CM, Huang Y, Meyer JG, Pankratz VS, Goode EL: Performance of amplified DNA in an Illumina GoldenGate BeadArray assay. Cancer epidemiology biomarkers prevention a publication of the American Association for Cancer Research cosponsored by the American Society of Preventive Oncology. 2008, 17: 1781-1789. 10.1158/1055-9965.EPI-07-2849.View ArticleGoogle Scholar
- Berthier-Schaad Y, Kao WHL, Coresh J, Zhang L, Ingersoll RG, Stephens R, Smith MW: Reliability of high-throughput genotyping of whole genome amplified DNA in SNP genotyping studies. Electrophoresis. 2007, 28: 2812-2817. 10.1002/elps.200600674.View ArticlePubMedGoogle Scholar
- Usdin K, Woodford KJ: CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro. Nucleic Acids Res. 1995, 23: 4202-4209. 10.1093/nar/23.20.4202.PubMed CentralView ArticlePubMedGoogle Scholar
- McDowell DG, Burns NA, Parkes HC: Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR. Nucleic Acids Res. 1998, 26: 3340-3347. 10.1093/nar/26.14.3340.PubMed CentralView ArticlePubMedGoogle Scholar
- Benita Y, Oosting RS, Lok MC, Wise MJ, Humphery-Smith I: Regionalized GC content of template DNA as a predictor of PCR success. Nucleic Acids Res. 2003, 31: e99-10.1093/nar/gng101.PubMed CentralView ArticlePubMedGoogle Scholar
- Baskaran N, Kandpal RP, Bhargava AK, Glynn MW, Bale A, Weissman SM: Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. 1996, 6: 633-638. 10.1101/gr.6.7.633.View ArticlePubMedGoogle Scholar
- Howell R, Usdin K: The ability to form intrastrand tetraplexes is an evolutionarily conserved feature of the 3’ end of L1 retrotransposons. Mol Biol Evol. 1997, 14: 144-155. 10.1093/oxfordjournals.molbev.a025747.View ArticlePubMedGoogle Scholar
- Simpson JR, Nagle WA, Bick MD, Belli JA: Molecular Nature of Mammalian Cell DNA in Alkaline Sucrose Gradients. Proc Natl Acad Sci USA. 1973, 70: 3660-3664. 10.1073/pnas.70.12.3660.PubMed CentralView ArticlePubMedGoogle Scholar
- Russell AP, Holleman DS: The thermal denaturation of DNA: average length and composition of denatured areas. Nucleic Acids Res. 1974, 1: 959-978. 10.1093/nar/1.8.959.PubMed CentralView ArticlePubMedGoogle Scholar
- Hollox EJ, Huffmeier U, Zeeuwen PLJM, Palla R, Lascorz J, Rodijk-Olthuis D, Van De Kerkhof PCM, Traupe H, De Jongh G, Den Heijer M, Reis A, Armour JAL, Schalkwijk J: Psoriasis is associated with increased beta-defensin genomic copy number. Nat Genet. 2008, 40: 23-25. 10.1038/ng.2007.48.PubMed CentralView ArticlePubMedGoogle Scholar
- Armour JAL, Palla R, Zeeuwen PLJM, Den Heijer M, Schalkwijk J, Hollox EJ: Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats. Nucleic Acids Res. 2007, 35: e19-10.1093/nar/gkl1089.PubMed CentralView ArticlePubMedGoogle Scholar
- Chakrabarti R, Schutt CE: The enhancement of PCR amplification by low molecular-weight sulfones. Gene. 2001, 274: 293-298. 10.1016/S0378-1119(01)00621-7.View ArticlePubMedGoogle Scholar
- Frackman BS, Kobs G, Simpson D, Storts D, Corporation P: Betaine and DMSO: Enhancing Agents for PCR. Promega Notes. 1998, 65: 9-12.Google Scholar
- Oshima RG: Single-stranded DNA binding protein facilitates amplification of genomic sequences by PCR. Biotechniques. 1992, 13: 188-PubMedGoogle Scholar
- Rees WA, Yager TD, Korte J, Von Hippel PH: Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry. 1993, 32: 137-144. 10.1021/bi00052a019.View ArticlePubMedGoogle Scholar
- Geiduschek EP: On the factors controlling the reversibility of DNA denaturation. J Mol Biol. 1962, 4: 467-487. 10.1016/S0022-2836(62)80103-X.View ArticlePubMedGoogle Scholar
- Landi MT, Chatterjee N, Yu K, Goldin LR, Goldstein AM, Rotunno M, Mirabello L, Jacobs K, Wheeler W, Yeager M, Bergen AW, Li Q, Consonni D, Pesatori AC, Wacholder S, Thun M, Diver R, Oken M, Virtamo J, Albanes D, Wang Z, Burdette L, Doheny KF, Pugh EW, Laurie C, Brennan P, Hung R, Gaborieau V, McKay JD, Lathrop M, et al, et al: A Genome-wide Association Study of Lung Cancer Identifies a Region of Chromosome 5p15 Associated with Risk for Adenocarcinoma. Am J Hum Genet. 2009, 85: 679-691. 10.1016/j.ajhg.2009.09.012.PubMed CentralView ArticlePubMedGoogle Scholar
- Diskin SJ, Li M, Hou C, Yang S, Glessner J, Hakonarson H, Bucan M, Maris JM, Wang K: Adjustment of genomic waves in signal intensities from whole-genome SNP genotyping platforms. Nucleic Acids Res. 2008, 36: e126-10.1093/nar/gkn556.PubMed CentralView ArticlePubMedGoogle Scholar
- Aird D, Ross MG, Chen W-S, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 2011, 12: R18-10.1186/gb-2011-12-2-r18.PubMed CentralView ArticlePubMedGoogle Scholar
- Blanco L, Bernad A, Lázaro JM, Martín G, Garmendia C, Salas M: Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem. 1989, 264: 8935-8940.PubMedGoogle Scholar
- Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC: Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet. 1998, 19: 225-232. 10.1038/898.View ArticlePubMedGoogle Scholar
- Hert DG, Fredlake CP, Barron AE: Advantages and limitations of next-generation sequencing technologies: a comparison of electrophoresis and non-electrophoresis methods. Electrophoresis. 2008, 29: 4618-4626. 10.1002/elps.200800456.View ArticlePubMedGoogle Scholar
- Brockman W, Alvarez P, Young S, Garber M, Giannoukos G, Lee WL, Russ C, Lander ES, Nusbaum C, Jaffe DB: Quality scores and SNP detection in sequencing-by-synthesis systems. Genome Res. 2008, 18: 763-770. 10.1101/gr.070227.107.PubMed CentralView ArticlePubMedGoogle Scholar
- Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, Boutell JM, Bryant J, Carter RJ, Keira Cheetham R, Cox AJ, Ellis DJ, Flatbush MR, Gormley NA, Humphray SJ, Irving LJ, Karbelashvili MS, Kirk SM, Li H, Liu X, Maisinger KS, Murray LJ, Obradovic B, Ost T, Parkinson ML, Pratt MR, et al: Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 2008, 456: 53-59. 10.1038/nature07517.PubMed CentralView ArticlePubMedGoogle Scholar
- Marioni JC, Thorne NP, Valsesia A, Fitzgerald T, Redon R, Fiegler H, Andrews TD, Stranger BE, Lynch AG, Dermitzakis ET, Carter NP, Tavaré S, Hurles ME: Breaking the waves: improved detection of copy number variation from microarray-based comparative genomic hybridization. Genome Biol. 2007, 8: R228-10.1186/gb-2007-8-10-r228.PubMed CentralView ArticlePubMedGoogle Scholar
- Staaf J, Vallon-Christersson J, Lindgren D, Juliusson G, Rosenquist R, Höglund M, Borg Å, Ringnér M: Normalization of Illumina Infinium whole-genome SNP data improves copy number estimates and allelic intensity ratios. BMC Bioinforma. 2008, 9: 409-10.1186/1471-2105-9-409.View ArticleGoogle Scholar
- Peiffer DA, Le JM, Steemers FJ, Chang W, Jenniges T, Garcia F, Haden K, Li J, Shaw CA, Belmont J, Cheung SW, Shen RM, Barker DL, Gunderson KL: High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping. Genome Res. 2006, 16: 1136-1148. 10.1101/gr.5402306.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.