Project description:Background: Insufficient quantities of human genomic DNA are a limiting factor for many clinical applications. Whole genome amplification (WGA) is an approach designed to overcome small amount of DNA for genome-wide genetic tests as it allows amplification of the entire genome from picogram or nanogram quantities of DNA. Various strategies of WGA have been developed; however, none of them can guarantee the absence of amplification bias. High-quality genome-representative amplified DNA is crucial for WGA use in basic research and clinical genetics. Thus, systematic evaluation of WGA effect on downstream methods is necessary. Results: In this paper, 4 multiple displacement amplification (MDA) -based and 2 PCR-based WGA kits were compared in their effect on segmental copy-number changes as well as copy-number neutral loss of heterozygosity detection by high-density oligonucleotide DNA arrays. We described outcomes and limits for each individual WGA; however, the main goal of this study was chiefly to show a general compatibility and features specific for particular WGA strategy. The main outcomes are as follows: 1) MDA-based WGAs showed higher tendency to generate false positive imbalances in contrast to PCR-based WGAs with higher risk of false negativity; 2) the specific risk of false positivity and/or negativity increased with decreasing copy-number segments size; 3) single-cell WGAs showed significantly worse effect on results in comparison to WGAs with nanogram level of DNA as input; 4) PCR-based WGAs were not compatible with copy-number neutral loss of heterozygosity analysis based on single nucleotide polymorphisms in restriction digestion sites and also showed higher risk of copy-number neutral loss of heterozygosity false negativity if combined with analysis based on simple hybridization. Conclusions: This study gives a comprehensive insight into the WGA effect on DNA array analysis. The results of this study help to choose WGA according to individual user requirements and options. Moreover, we show a strategy to verify and validate segmental copy-number changes detection by DNA array protocol including any WGA for any purpose to attain the highest efficiency without an unnecessary WGA bias.

Project description:Methods for haplotyping and DNA copy number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required that substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP-genotypes (AA, AB, BB), and DNA copy number profiling remains difficult as true DNA copy number aberrations have to be discriminated from WGA-artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by deciphering WGA-distorted SNP B-allele fractions, using a process we coin haplarithmisis. We demonstrate clinical precision of the method on single cells biopsied from human embryos to diagnose disease alleles genome wide, we advance and facilitate the detection of numerical and structural chromosomal anomalies in single cells, and can distinguish meiotic from mitotic segregation errors in a single assay. The samples of a reference family were applied for optimisation of single-cell genotyping using Affymetrix SNP-arrays prior to downstream analysis. Specifically, the reference family delivers genomic DNA samples isolated from peripheral blood of two siblings 'S1' and 'S2', the mother and father of these siblings, as well as of the maternal grandmother and grandfather. Of individuals ‘S1’ and ‘S2’, six EBV-transformed lymphoblastoid single cells were isolated of which three were whole-genome amplified using MDA and three using PicoPlex. These WGA-products were hybridized to Affymetrix NspI 250K SNP-arrays following the protocol as recommended by the company. Subsequently, the SNP-probe signals were interpreted by different genotyping algorithms (see data processing). Based on overall performance, it was decided to use the Dynamic Model (DM) for interpreting Affymetrix SNP-probe signals of single cells.

Project description:In the study of tumor genetics, formalin-fixed paraffin-embedded (FFPE) tumors are the most readily available tissue samples. While DNA derived from FFPE tissue has been validated for array comparative genomic hybridization (aCGH) application, the suitability of such fragmented DNA for single-nucleotide polymorphism (SNP) array analysis has not been well examined. Furthermore, whole-genome amplification (WGA) has been used in the study of small precursor lesions to produce sufficient amount of DNA for aCGH analysis. It is unclear whether the same approach can be extended to SNP analysis. In this study, we examined the utility and limitations of genotyping platform performed on whole-genome amplified DNA from FFPE tumor samples for both copy number and SNP analyses. We analyzed the results obtained using DNA derived from matched FFPE and frozen tissue samples on Affymetrix 250K Nsp SNP array. Two widely used WGA methods, Qiagen (isothermal protocol) and Sigma (thermocycling protocol), were used to determine how WGA methods affect the results. We found that the use of DNA derived from FFPE tumors (without or with WGA) for high-resolution SNP array application can produce a significant amount of false positive and false negative findings. While some of these misinterpretations appear to cluster in genomic regions with high or low GC contents, the majority appears to occur randomly. Only large-scale chromosome LOH (>10Mb) can be reliably detected from FFPE tumor DNA samples (without or with WGA) but not smaller LOH or copy number alterations. Our findings here indicate a need for caution in SNP array data interpretation when using FFPE tumor-derived DNA, particularly with WGA. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cryopreserved and FFPE mesenchymal tumor samples without or with WGA, as well as genomic snap-frozen non-neoplastic tissue DNA from 5 adult individuals to serve as reference DNA. WGA was performed using the REPLI-g® FFPE kit (Qiagen, Valencia, CA, USA) and GenomePlex® Tissue Whole Genome Amplification WGA5 kit (Sigma, Saint Louis, MO, USA) in parallel in accordance with the manufacturers’ protocols. A two- to eight-hour individualized reaction time was used in the Qiagen platform for each sample. A gradient amount of initial DNA (10ng, 30ng, 60ng, 100ng and 150ng) was tested followed by gel electrophoresis and qualitative multiplex PCR assay to determine the quality of post-WGA products. At least four independent experiments were concurrently performed per template amplification. Four separateWGA reaction products were pooled for each sample for subsequent microarray analysis to minimize the amplification bias and allele dropout. One of the Affymetrix GeneChip® Human Mapping 500K Array Set (Nsp 250K SNP array) was used for genotyping analysis. Four gastrointestinal stromal tumors with known cytogenetic aberrations were included. Two cases were sucessfullly amplified and passed the quality tests. A total of 12 samples were compared between each other, including frozen tissue DNA (as reference), frozen tissue DNA with WGA (two platforms), FFPE tissue DNA, and FFPE tissue DNA with WGA (two platforms) from each case.

Project description:Methods for haplotyping and DNA copy number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required that substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP-genotypes (AA, AB, BB), and DNA copy number profiling remains difficult as true DNA copy number aberrations have to be discriminated from WGA-artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by deciphering WGA-distorted SNP B-allele fractions, using a process we coin haplarithmisis. We demonstrate clinical precision of the method on single cells biopsied from human embryos to diagnose disease alleles genome wide, we advance and facilitate the detection of numerical and structural chromosomal anomalies in single cells, and can distinguish meiotic from mitotic segregation errors in a single assay. Here we provide two sample sets, including (1) a reference family delivering samples applied for the development and optimization of single-cell genotyping, QC-metrics, haplarithmisis and the siCHILD algorithm. Specifically, the reference family delivers genomic DNA samples isolated from peripheral blood of two siblings 'S1' and 'S2', the mother and father of these siblings, as well as of the maternal grandmother and grandfather. Of individuals ‘S1’ and ‘S2’, six EBV-transformed lymphoblastoid single cells were also isolated, of which three were whole-genome amplified using MDA and three using PicoPlex. These multi-cell genomic DNA samples and single-cell WGA-products were hybridized to Illumina HumanCytoSNP12-v2.1 SNP-arrays following: (i) the protocol as recommended by the company and/or (ii) a modified rapid protocol as described below. Subsequently, the SNP-probe signals were interpreted by two different genotyping algorithms (GenCall and GenoSNP). Based on overall performance, we decided to use GenCall for interpreting Illumina SNP-probe signals of single-cell and multi-cell DNA samples. (2) A set of 12 families undergoing genetic diagnosis (for Mendelian disorders or translocation chromosomes) of preimplantation embryos following in vitro fertilization. In general, each family delivers genomic DNA samples isolated from peripheral blood of the two parents as well as a sibling and/or other close relatives; the specific kinships for each family are given in the 'description' column. In addition, of each family single blastomeres biopsied from preimplantation embryos obtained via in vitro fertilization of the parental gametes are also provided. All single-cell genomes were amplified by MDA. All the samples were hybridized to Illumina HumanCytoSNP12-v2.1 SNP-arrays following the rapid protocol. This data was used for clinical validation of the siCHILD algorithm.

Project description:Establishment and subsequent validation of a aCGH protocol for WGA (whole genome amplification) products originating from single cell or low amount of starting material (i.e. microdissected FFPE tissue samples). The establishment of the protocol involved testing of three DNA labeling protocols. Two labeling protocols were designed specifically for Ampli1(TM) WGA products. Additionally random primed isothermal (Klenow-based) labeling approach was tested (MM-CM-6hlendick et al., PLoS One. 2013 Jun 25;8(6):e67031.). In addition two different types of reference samples were tested and reference based on single-cell WGA products was chosen as most suitable in the end. The validation of the protocol assessed the following aspects: (1) performance of the protocol on primary and reamplified WGA products, (2) accuracy of the protocol in term of sensitivity of the CNA detection, (3) accuracy in terms of recapitulation of complex patterns of CNAs, (4) accuracy in terms of quantitative assessment of the CNAs, (5) ability to detect genomic heterogeneity of single cells (obtained either from in vitro cultures or from clinical patient material), (6) ability to detect minimal regions of aberration within a panel of disseminated cancer cells and corresponding tumor tissues. Establishment and validation of the single-cell aCGH protocol: two condition experiment (i.e. PCR-based labeling technique 1 vs. PCR-based labeling technique 2; PCR-based labeling technique 2 vs. random-primed isothermal (Klenow) labeling approach; reference DNA from cell pool WGA product vs. reference DNA from single-cell WGA products). Validation of the protocol: comparison of the CNA profiles between single-cell WGA products and corresponding bulk DNA. Analysis of the DCC and corresponding FFPE tumor tissue samples: single-condition experiment performed on samples collected at different stages of the disease (DCCs from bone marrow) and/or from different sites (primary tumor-breast; metastasis-lymph nodes; DCCs-bone marrow). Microarray data is corresponding data depicted in the paper manuscript titled: Reliable single cell array CGH for clinical samples

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Single Cell Whole Genome Amplification Technique Impacts the Accuracy of SNP Microarray Based Genotyping and Copy Number Analyses

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