Project description:Illumina Infinium whole genome genotyping (WGG) arrays are increasingly being applied in cancer genomics to study gene copy number alterations and allele-specific aberrations such as loss-of-heterozygosity (LOH). Methods developed for normalization of WGG arrays have mostly focused on diploid, normal samples. However, for cancer samples genomic aberrations may confound normalization and data interpretation. Therefore, we examined the effects of the conventionally used normalization method for Illumina Infinium arrays when applied to cancer samples. We demonstrate an asymmetry in the detection of the two alleles for each SNP, which deleteriously influences both allelic proportions and copy number estimates. The asymmetry is caused by a remaining bias between the two dyes used in the Infinium II assay after using the normalization method in Illumina’s proprietary software (BeadStudio). We propose a quantile normalization strategy for correction of this dye bias. We tested the normalization strategy using 535 individual hybridizations from 10 data sets from the analysis of cancer genomes and normal blood samples generated on Illumina Infinium II 300k version 1 and 2, 370k and 550k BeadChips. We show that the proposed normalization strategy successfully removes asymmetry in estimates of both allelic proportions and copy numbers. Additionally, the normalization strategy reduces the technical variation for copy number estimates while retaining the response to copy number alterations. The proposed normalization strategy represents a valuable low-level analysis tool that improves the quality of data obtained from Illumina Infinium arrays, in particular when used for LOH and copy number variation studies. To investigate the effects of a quantile normalization of Illumina Infinium data, compared to conventional normalization using BeadStudio (www.illumina.com), we renormalized 535 individual hybridizations conducted on Illumina 300K, 370K and 550K BeadChips. Sample types included breast cancer, colon cancer, urothelial carcinoma, leukemia as well as normal blood and HapMap samples. This series includes the 6 breast cancers hybridized on Illumina HumanHap 550K BeadChips.

Project description:We applied whole-genome single nucleotide polymorphism (SNP) arrays to define a comprehensive genetic profile of 23 esophageal adenocarcinoma (EAC) primary tumor biopsies based on loss of heterozygosity (LOH) and DNA copy number changes. Alterations were common, averaging 97 (range 23-208) per tumor. LOH and gains averaged 33 (range 3-83) and 31 (range 11-73) per tumor, respectively. Copy neutral LOH events averaged 27 (range 7-57) per EAC. We noted 126 homozygous deletions (HDs) across the EAC panel (range 0-11 in individual tumors). Frequent HDs within FHIT (17/23), WWOX (8/23) and DMD (6/23) suggest a role for common fragile sites or genomic instability in EAC etiology. HDs were also noted for known tumor suppressor genes (TSGs) including: CDKN2A, CDKN2B, SMAD4 and GALR1, and identified PDE4D and MGC48628 as potentially novel TSGs. All tumors showed LOH for most of chromosome 17p, suggesting that TSGs other than TP53 maybe targeted. Frequent gains were noted around MYC (13/23), BCL9 (12/23), CTAGE1 (14/23) and ZNF217 (12/23). Thus, we have confirmed previous reports indicating frequent changes to FHIT, CDKN2A, TP53 and MYC in EAC and identified additional genes of interest. Meta analysis of previous genome-wide EAC studies together with the data presented here, highlighted consistent regions of gain on 8q, 18q and 20q, and multiple LOH regions on 4q, 5q, 17p and 18q, suggesting that more than one gene may be targeted on each of these chromosome arms. The focal gains and deletions documented here are a step towards identifying the key genes involved in EAC development. Keywords: High Density SNP array Here we use Illumina 317K whole-genome single-nucleotide polymorphism arrays to define a comprehensive allelotype of melanoma based on loss of heterozygosity (LOH) and copy number changes in a panel of 23 esophageal adenocarcinoma (EAC) primary tumor biopsies. Each EAC was paired to normal squamous biopsy from 40328 (GSM266078) to generate the log_R_ratio.

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.

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: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.

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Genome Alteration Print (GAP): a tool to visualize and mine complex cancer genomic profiles obtained by SNP-arrays

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Genome Alteration Print (GAP): a tool to visualize and mine complex cancer genomic profiles obtained by SNP arrays.

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