Speicherlab-Human-PlasmaDNA-CGH
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
DISEASE(S): Colon Cancer
SUBMITTER: Peter Ulz
PROVIDER: E-MEXP-3416 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:About 230 clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but no assays enable comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for CTC enrichment and isolation with 100% purity with a non-random whole genome amplifiation method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 breast cancer patients. We defined a genome integrity index (GII) to identify cells suited for molecular chracterization by different molecular assay in more than 90% of single cells, such as diagnostic profiling for point mutations, gene amplifications and whole genomes of single cells. The high reliability on clinical samples enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified therapeutically relevant genomic disparity between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered preexisting cells reistsant to ERBB2 targeted therapies suggesting ongoing microevolution at late stage disease whose exploration may provide essential information for personalized treatment decisions. The analysis aimed to indentify profiles of copy number changes in genomic DNA of single circulating tumor cells (CTCs). For this, CTCs were enrched using the FDA approved CellSearch System and single-cell were isolated using the DEPArray System. Subsequently, single-cell DNA was amplified using the Ampli1 WGA Kits and subjected to single-cell aCGH analysis according to previously published protocol (Czyz ZT et al., PLoS One. 2014 Jan 21;9(1):e85907). The analysis included 38 single CTCs and 10 white blood cells (WBCs) obtained from 16 breast cancer patient. WBCs were used as controls for the analysis. In addition, four CTC cell pools were included in the analysis. This was done to show the discrepancies between the profiles of individual cells and corresponing average genomic profile of CTCs in a patient material (cell pools), thereby demonstrating the importance of the analysis on the cell-by-cell basis. The reference sample used for all aCGH experiments consisted of a pool of four single-cell WGA products generated from WBCs of a healthy female donor.
2014-11-05 | E-GEOD-58192 | biostudies-arrayexpress
Project description:Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), were validate with Illumina HumanHap550-Duo Genotyping Beadchip. As seen in (Suppl. Table 5a), the call rates (97.30% to 99.07%) and accuracy or concordance ( > 99.85% for the SNPs called in both amplicon and natural reference) for 5 ng derived amplicons with both Wpa and Gv2 were close to each other and close to native gDNA (call rate: 98.3% to 99.75%). These call rates were better than a recent report (amplicon 95.9% vs. un-amplified 98.5%), in which the early kit Repli-g 625S was applied, and re-genotyping was performed when the performance was low and duplicate samples were filtered for the highest call rate. The genotyping accuracy of Wpa was actually in the same range as the variation in technical replicates with similar SNP typing arrays (99.87% and 99.88%, replicated Affymetrix array, or between Affymetrix and Illumina arrays). Importantly, the genotyping concordance for amplicons generated from 0.5 ng with Wpa (99.88% and 99.69%) were also close to the technical replicates. In this case, the call rates of Wpa were slightlyreduced compared to that with 5 ng input, but the call rate for the partially degraded sample 04-018, was modestly improved over Gv2 (92.06 % vs. 90.53%). Wpa data also showed some amplification non-uniformity among different locations, resulting in some âartificial CNVsâ similar to Gv2 (exampled as in Suppl. Fig. 5 and Suppl. Table 6), with the outputs obtained by taking unamplified gDNAs as their reference. This imbalance however was consistent and reproducible for each method but different between Wpa and Gv2. These artificial CNVs can be efficiently cancelled if pair-wise amplified test and reference are compared, as observed in CGH result (Fig. 4 and Suppl. Fig. 4), also supported by others {Pugh 2008}. It is interesting to note that the representation of chromosomal terminal sequences was greatly improved with Wpa compared with Gv2 (Fig. 5), and that some of these regions were significantly under-amplified or even lost with Gv2 (Suppl. Fig. 5 and Suppl. Table 6, 7), as also independently reported recently {Pugh 2008}. This occurred especially in the terminal 3 to 5 Mb and sometimes extended to 10 Mb in many chromosome termini, and was particularly serious when low levels or degraded DNA was as input. An analysis for 5 Mb termini is shown (Suppl. Table 5b calculated all involved SNPs as a cohort. Fig. 5 and Suppl. Tables 6 and 7 were the result for each chromosome terminus). Importantly, the SNP typing was also greatly improved, outstandingly exemplified by the amplicons of 0.5 ng input for the partially degraded 04-018, with Wpa versus Gv2 call rate of 91.9% vs. 84.45% and accuracy of 99.57% vs. 98.62%. The result also showed that these terminal regions underrepresentation in Gv2 was not absolutely associated with the distance-to-end, but possibly was a sequence related issue. Keywords: Whole-pool amplification, whole genome SNP typing The overall goal of the part of study was a validation of the quality of the amplicons from different amounts (5ng and 0.5 ng) of original starting gDNA, good quality (sample 05-050) or partially degraded gDNA (sample 04-018), with our new procedure Wpa, and with native gDNA as control, in terms of the call rate and accuracy (allele bias) in addition to the uniformity of the sequence amplified (sequence representation or sequence bias). Amplified or native genomic DNA isolated from patients was in-parallel analyzed/genotyped with the same experimental platform, of which the native genomic DNAs were used as the standard controls. For the sequence representation, the two alleles of the SNPsâ signal of a panel of multiple native DNAsâ signal provided by the experimental platform (Illumina) was used as the reference, so that an abstract signal for sequence representation of each SNP and for all SNPs was obtained.
2010-06-25 | E-GEOD-12732 | biostudies-arrayexpress
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2009-02-24 | E-MEXP-1806 | biostudies-arrayexpress
Project description:Wild type, elg1d, pol30f, and pol30f elg1d cells were arrested in G1 and synchronously released in the presence of HU for 30 or 60 minutes. Genomic DNA was extracted, amplified, and hybridized to a whole genome tiling microarray. Replicated regions were identified as peaks that exhibit increased copy number relative to G1 cells and overlapped confirmed replication origins.
2011-12-20 | E-MEXP-3458 | biostudies-arrayexpress
Project description:Background Urothelial carcinoma of the bladder (UC) is a common malignancy. Although extensive transcriptome analysis has provided insights into the gene expression patterns of this tumor type, the mechanistic underpinnings of differential methylation remain poorly understood. Multi-level genomic data may be used to profile the regulatory potential and landscape of differential methylation in cancer and gain understanding of the processes underlying epigenetic and phenotypic characteristics of tumors. Methods We perform genome-wide DNA methylation profiling of 98 gene-expression subtyped tumors to identify between-tumor differentially methylated regions (DMRs). We integrate multi-level publically available genomic data generated by the ENCODE consortium to characterize the regulatory potential of UC DMRs. Results We identify 5,453 between-tumor DMRs and derive four DNA methylation subgroups of UC with distinct associations to clinicopathological features and gene expression subtypes. We characterize three distinct patterns of differential methylation and use ENCODE data to show that tumor subgroup-defining DMRs display differential chromatin state, and regulatory factor binding preferences. Finally, we characterize an epigenetic switch involving the HOXA-genes with associations to tumor differentiation states and patient prognosis. Conclusions Genome-wide DMR methylation patterns are reflected in the gene expression subtypes of UC. UC DMRs display three distinct methylation patterns, each associated with intrinsic features of the genome and differential regulatory factor binding preferences. Epigenetic inactivation of HOX-genes correlates with tumor differentiation states and may present an actionable epigenetic alteration in UC. MeDIP hybridizations on 98 human urothelial carcinoma samples and 4 normal urothelium samples on Nimblegen 3x720K RefSeq Promoter and CGI aCGH arrays.
2015-05-28 | E-GEOD-58256 | biostudies-arrayexpress
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