Project description:We investigated the influence of genome position on propensity to amplify. First, we integrated a mutant form of DHFR into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells. We observed site specific differences in methotrexate sensitivity, organization of amplicons and amplification frequency. One site was uniquely associated with a significantly enhanced propensity to amplify and recurrent amplicon boundaries, possibly implicating a rare folate sensitive fragile site in initiating amplification. Hierarchical clustering of gene expression patterns and subsequent gene enrichment analysis revealed two clusters differing significantly in expression of MYC target genes independent of integration site. We introduced a mutant form of DHFR (L22F), which confers greater resistance to methotrexate than the wild type (endogenous) gene into HCT116+chr3 cells and isolated independent clones containing DHFR* at different positions in the genome and identified genome sequences flanking the integration site of DHFR* using inverse PCR. For further analysis, we selected only clones, which were considered to have a single insertion of DHFR* by inverse PCR (13 independent insertion sites). The individual insertion site clones were further characterized with respect to genome copy number profiles. To select methotrexate resistant colonies, we exposed cells to a concentration of methotrexate that was three to four times the IC-50 for each integration site. Genomic copy number profiles were obtained for isolated resistant colonies (GSE6262) by using UCSF HumArray platform (GPL4421). Twelve methotrexate resistant colonies (four different integration sites) were selected for microarray analysis of gene expression at the mRNA level. The untreated integration site clone was used as the reference (Cy5 labeled cDNA) for each of the hybridizations with its respective resistant colonies (Cy3 labeled cDNA). Hybridizations were carried out on arrays of printed long oligonucleotides (70mers) containing 21,000 elements (Operon V2.0, printed in J. David Gladstone Institutes, Genomics Core Laboratory).

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), faithfully demonstrated all previously known heterozygous segmental duplications and deletions (3 Mb to 18 kb) located on chromosome 22 and even a homozygous deletion smaller than 1 kb with high resolution chromosome-wide CGH. Specifically, HR-CGH with 5 ng-input gDNA-derived amplicon detected all previously known chromosomal segmental aberrations in chromosome 22 in samples from two different probands, and was indistinguishable from the HR-CGH result with native gDNA from the same probands (Fig. 4, Fig. S3 and S4). The break points were also precisely demonstrated. These include a heterozygous genomic segmental duplication (3 copies, 3 Mb in size, sample 05-050, Fig. 4) and 2 different heterozygous deletions (1 copy, 1.4 Mb and 18 kb respectively, sample 04-018, Fig. S4), all of which are located in or bounded by regions of low copy repeats (LCRs). In addition, a previous known homozygous deletion of 975 bp (in 04-018 and 05-050) was again accurately demonstrated (05-050 data showed in Fig. 4c), although sometimes (04-018) the data was a little noisier than with unamplified DNA (Fig. S4d). In contrast, the Wpa-40oC resulted in abundant signal noise and failed in detection of these copy number aberrations (Fig. 4, Fig. S3, S4). Impressively, HR-CGH with 0.5 ng gDNA-derived amplicons via Wpa also clearly detected the known CNVs, although noisier (Fig. S4). The 0.1ng gDNA derived amplicons via Wpa could not unambiguously show CNVs because of higher variability of signals, but the CNVs’ patterns were mostly well maintained (Fig. S4 for 04-018). We did also notice some locus-imbalance in the amplicon, however this was minimized, and was reproducible when the input was above a certain threshold amount, and it could be well compensated if the same amplified reference sample was applied in parallel as showed above. Keywords: Whole-pool amplification, high resolution comparative genome hybridization (HR-CGH)

Project description:A summary of the work associated to these microarrays is the following:; Methotrexate (MTX) is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we have performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the DHFR gene or as a result of a transcriptome screening using microarrays. Genes adjacent to dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCa genes in treated cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. We provide functional evidences indicating that caveolin 1 and E-cadherin may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy. Experiment Overall Design: Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M MTX. Six samples are provided which correspond to triplicated of each cell line. The samples provided were subsequently normalyzed and analyzed using the specific software GeneSpring GX v7.3.1.

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 “artificial CNVs” 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

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.

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Genome position and gene amplification - higher resolution mapping of amplicons

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