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

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.

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MetaHit Single Cell WGA

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