Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays (original) (raw)
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Microarray analysis of copy number variation in single cells
Nature Protocols, 2012
amplified Dnas of a cell are hybridized to a 3,000-bacterial artificial chromosome (Bac) array and to an affymetrix 250,000 (250K)-snp array. subsequent copy number calling is based on the integration of Bac probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGa) reference model for diploid chromosomes, as well as snp copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). all methods for detecting Dna copy number aberrations in single human cells have difficulty in confidently discriminating WGa artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental Dna imbalance detection. our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGa samples. the protocol takes 5-7 d. protocol 282 | VOL.7 NO.2 | 2012 | nature protocols 50K XbaI Affymetrix Affymetrix CNAT Highest resolution was not mentioned 14 (continued)
PLoS ONE, 2013
Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4,9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at basepair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (.1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.
High resolution array-CGH analysis of single cells
Nucleic Acids Research, 2007
Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (Genome-Plex Ò Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader-Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics.
Molecular Human Reproduction, 2009
The scarce amount of DNA contained in a singe cell is a limiting factor for clinical application of PGD mainly due to the risk of misdiagnosis caused by allele dropout and the difficulty in obtaining copy number variations in all 23 pairs of chromosomes. Multiple displacement amplification (MDA) has been reported to generate large quantity of products from small amount of templates. Here, we evaluated the fidelity of whole genome amplification MDA from single or a few cells and determined the accuracy of chromosome copy number assessment on these MDA products using an Affymetrix 10K 2.0 SNP Mapping Array. An average coverage rate (86.2%) from single-cells was obtained and the rates increased significantly when 5 or more cells were used as templates. Higher concordance for chromosome copy number from single cells could be achieved when the MDA amplified product was used as reference (93.1%) than when gDNA used as reference (82.8%). The present study indicates that satisfactory genome coverage can be obtained from single cell MDA which may be used for studies where only a minute amount of genetic materials is available. Clinically, MDA coupled with SNP mapping array may provide a reliable and accurate method for chromosome copy number analysis and most likely for the detection of single gene disorders as well.
Scientific reports, 2017
The growing interest in liquid biopsies for cancer research and cell-based non-invasive prenatal testing (NIPT) invigorates the need for improved single cell analysis. In these applications, target cells are extremely rare and fragile in peripheral circulation, which makes the genetic analysis very challenging. To overcome these challenges, cell stabilization and unbiased whole genome amplification are required. This study investigates the performance of four WGA methods on single or a limited number of cells after 24 hour of Streck Cell-Free DNA BCT preservation. The suitability of the DNA, amplified with Ampli1, DOPlify, PicoPLEX and REPLI-g, was assessed for both short tandem repeat (STR) profiling and copy number variant (CNV) analysis after shallow whole genome massively parallel sequencing (MPS). Results demonstrate that Ampli1, DOPlify and PicoPLEX perform well for both applications, with some differences between the methods. Samples amplified with REPLI-g did not result in s...
Biotechnology & Biotechnological Equipment, 2011
Array-based comparative genomic hybridization (array CGH) is a powerful and high-resolution approach for detection of DNA copy number gains and losses, associated to chromosomal abnormalities, many of which are below the detection limit of the routine cytogenetic analysis. Technological improvements to acquire higher resolution have led to generation of novel microarray platforms harboring larger numbers of shorter DNA sequences (oligonucleotides). Two main technologies are currently used for DNA copy number genome profiling -the Bacterial Artificial Chromosome and the oligo-based CGH arrays. In this study we present a comparison of two array-based platforms for genome-wide copy number screening in a group of six patients with idiopathic congenital anomalies and mental retardation or developmental delay. Each of our patients was studied separately with BAC and oligo-based microarray and the imbalances found were FISH confirmed. Our results indicated that the oligo-based platform had higher sensitivity than the BAC arrays, which allowed more precise detection of microdeletion/microduplication changes and could also improve the quality of array CGH analysis for the purpose of clinical diagnosis.
Scientific Reports, 2017
Whole genome amplification (WGA) has become an invaluable tool to perform copy number variation (CNV) detection in single, or a limited number of cells. Unfortunately, current WGA methods introduce representation bias that limits the detection of small CNVs. New WGA methods have been introduced that might have the potential to reduce this bias. We compared the performance of PicoPLEX DNA-Seq (Picoseq), DOPlify, REPLI-g and Ampli-1 WGA for aneuploidy screening and copy number analysis using shallow whole genome massively parallel sequencing (MPS), starting from single or a limited number of cells. Although the four WGA methods perform differently, they are all suited for this application.
BMC genomics, 2018
The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations. By analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was...
Whole genome amplification (WGA) has become an invaluable tool to perform copy number variation (CNV) detection in single, or a limited number of cells. Unfortunately, current WGA methods introduce representation bias that limits the detection of small CNVs. New WGA methods have been introduced that might have the potential to reduce this bias. We compared the performance of PicoPLEX DNA-Seq (Picoseq), DOPlify, REPLI-g and Ampli-1 WGA for aneuploidy screening and copy number analysis using shallow whole genome massively parallel sequencing (MPS), starting from single or a limited number of cells. Although the four WGA methods perform differently, they are all suited for this application. Whole genome amplification (WGA) has become an invaluable tool to perform massively parallel sequencing (MPS) in applications where only a limited number of cells is available. Well-known examples of such applications are: preimplantation genetic diagnosis (PGD), cell-based non-invasive prenatal testing (NIPT) 1 and liquid biopsy of circulating tumor cells (CTCs) 2–4. Several WGA methods exist to amplify DNA extracted from a limited number of cells, yielding the necessary amount of DNA required to perform MPS 5, 6. The different WGA methods each have their advantages and disadvantages in terms of genome coverage, representation bias, error rates, yield and robustness. The best WGA method per se does not exist, because the downstream application is also important to determine the ideal method 5. Some WGA methods have already been compared for specific applications 5, 7–11. Multiple displacement amplification (MDA) methods are better suited for single nucleotide polymorphism (SNP) detection while PCR-based methods are the better option for copy number variant (CNV) detection 5. MDA methods use the high-fidelity phi29 polymerase, reducing nucleotide errors in the amplified sequences, while PCR-based methods tend to give a more uniform amplification across the genome. For the detection of CNVs, SurePlex WGA has proven its efficiency in clinical settings such as PGD 9, 12, 13. However, results show that the WGA representation bias is still a limiting factor hampering higher resolution copy number profiles when starting from a single or a limited number of cells 9. Fortunately, new WGA methods are being introduced that might have the potential to reduce this bias. In this study, we compared four different commercially available WGA methods for their suitability to detect CNVs after MPS. A first method, REPLI-g single cell WGA, is a well-established MDA method 5, 14. In a study comparing 5 different WGA methods, REPLI-g had the lowest false positive rate and was well-suited for detection of CNVs and single nucleotide variations (SNVs) 14. This two-step protocol includes an amplification step of 8 h, but the hands-on time is short. A second method, Ampli-1 WGA (Silicon Biosystems, Castel Maggiore, Italy), has already proven its efficiency for CNV and STR analysis in prenatal diagnosis 1. Ampli-1 is based on a ligation-mediated PCR following a site-specific DNA digestion. Usage of non-random primers is one of the factors leading to a more homologous coverage. Unfortunately, the protocol consists of many different steps and is time consuming. PicoPLEX DNA-Seq (Picoseq) (Rubicon Genomics Inc., MI 48108, USA) and DOPlify WGA (Reproductive Health Science, Thebarton, Australia) are the two most recently developed methods. Picoseq is a method based on the PicoPLEX/SurePlex WGA technology (Rubicon Genomics Inc., MI 48108,