Unknown,Transcriptomics,Genomics,Proteomics

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Affymetrix SNP 6.0 array data for human embryonic stem cell lines

ABSTRACT: Pluripotent human embryonic stem cells are studied for potential applications in regenerative cell replacement therapies because of their untransformed nature and unique capacity to self-renew and differentiate. However it is known that adaptation of hESCs occurs on prolonged culture and may be associated with the acquisition of chromosome abnormalities. We report a high resolution DNA SNP 6.0 array analysis showing copy number variation in 17 different hESC lines maintained in different laboratories. Several of the changes were culture induced and changed the expression levels of affected genes. Loss of heterozygosity (LOH) of chromosome 16q was detected in one line, in addition to several smaller LOH regions. Importantly, hESCs were found to contain several CNVs less than 3 Mb in size which is below the detection limit of conventional karyotyping. Copy number analysis of Affymetrix SNP 6.0 arrays was performed for 29 hESC samples of various passages and laboratory origin. 40 reference HapMap samples from Affymetrix were hybridized at the same time to built a reference genome used in the analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Elisa Närvä

PROVIDER: E-GEOD-15096 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We analysed, by last-generation high-resolution SNP arrays, Normal Karyotype (NK)-AML patients at diagnosis (Dx) and remission (R) phases, in order to determine the number of tumor-associated copy number abnormalities (CNAs) and copy neutral-loss of heterozygosity (CN-LOH) regions per patient and to identify possible recurring genomic abnormalities. The number of tumor-associated CNAs was detemined after comparison of 11 matched Dx/R samples using stringent conditions able to reduce the number of false positive CNAs. 8 additional unmatched Dx samples were included in the analysis of recurring CNAs and for detection of broad CN-LOH segments. With the exception of a single outlier case, a low number of CNAs per patient was detected (median value of 1 somatic loss or gain per patient). However, a high prevalence of CNAs (60-70% of the patients showed at least 1 tumor-associated gain or loss) and few recurring CNAs were observed, thus providing new hints towards identification of cooperating mutations. An extensive search of all CN-LOH regions > 1 Mb revealed only 3 broad regions (terminal 12Mb of 22q, terminal 27Mb of 1p and the whole chromosome 21) in three patients out of 19 (16%). CN-LOH of the whole chromosome 21 was responsible for homozygosity of a missense mutation (R80C) of RUNX1/AML1. Our study suggests that a relative submicroscopic copy number stability NK-AML genomes is associated with low recurrence of specific CNAs and CN-LOH in NK-AML patient population. Nineteen AML bone marrow samples were collected at the time of diagnosis (>90% blasts), defined as karyotypically-normal on the basis of standard metaphase cytogenetics (MC) and analysed by the Affymetrix SNP 6.0 arrays. In 11 of such cases (matched Dx) we obtained a bone marrow sample at the remission phase (matched R) of comparable high quality as evaluated by Contrast QC and MAPD values. In 8 cases (unmatched Dx) the corresponding R sample was not available or was not of comparable quality. As a reference set the publicly available data from 270 HapMap individuals were used.

Project description:Generating sufficient DNA for high-throughput genetic analysis has always been a challenge for clinical settings where the amount of source DNA is limited. Multiple displacement amplification (MDA) has been proposed as a promising candidate for such situations. Previous work with lower-resolution arrays confirmed the utility of single-cell MDA products for large-size (~30 Mb) genome variation screening. We tested the performance of single-cell MDA products on the SNP 6.0 arrays to examine the performance of single-cell MDA in SNP genotyping, copy number polymorphism, de novo copy number variation (CNV) and loss of heterozygosity (LOH) analysis. Our data show that for SNP genotyping, single-cell MDA did not obtain complete genome coverage or high sequence fidelity. For CNV calling, single-cell MDA introduced stochastic amplification artifacts in log2 ratio profiles, reducing the robustness of CNV calling; however, by adjusting smooth window size, it is still possible to analyze large chromosomal aberrations, and homozygous deletions as small as 500 kb can still be identified from the noisy log2 ratio profiles. Our results also suggest that even with a modified protocol (reduction of reaction volume, addition of a molecular crowding reagent, minimization of reaction time), single-cell MDA presented little improvement over the unmodified protocol, but by increasing the number of cells as template to 5M-bM-^@M-^S10 cells, SNP 6.0 array results comparable to those of 10 ng genomic DNA MDA could be obtained. Algorithms like PICNIC improved the CNV calling, suggesting that better algorithms can better utilize single-cell MDA array results. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cell line samples, and multiple displacement samples. Genotyping, Copy number and LOH analysis of Affymetrix SNP 6.0 arrays was performed for 3 samples of unamplified cell line genomic DNA, 2 samples of DNA obtained by multiple displacement amplification from 10ng genomic DNA, 3 single-cell multiple displacement amplification (MDA) products, single cell modified MDA amplification product, 5-cell modified MDA amplification product, 10-cell modified MDA amplification product.

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Affymetrix SNP 6.0 array data for human embryonic stem cell lines

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