Assessment of Whole Genome Amplification for Sequence Capture and Massively Parallel Sequencing

Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.     

Decreased mitochondrial DNA mutagenesis in human colorectal cancer

Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C:G to T:A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage.     

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.     

全基因组扩增策略在基因芯片分析SARS冠状病毒实验中的应用

针对SARS冠状病毒的分子生物学检测是控制SARS流行的关键环节。为评价全基因组扩增对SARS微量样本检测的影响 ,采用 6 mer随机引物反转录 ,用加接头的随机引物合成第二链 ,再以接头序列为引物扩增并掺入荧光标记 ,最后与带有 70 mer探针的基因芯片杂交。此非特异方法基本覆盖了样本中的全部DNA ,结果发现SARS冠状病毒全基因组的扩增效果对基因芯片杂交结果的均匀性有较大影响 ,PCR循环次数增多会导致扩增均匀性的降低。分析了不同的引物对全基因组扩增均匀性的影响 ,探讨了全基因组扩增策略的缺陷。     

Somatic mitochondrial DNA mutations in neurofibromatosis type 1-associated tumors

Neurofibromatosis type 1 is an autosomal dominantly inherited disease predisposing to a multitude of tumors, most characteristically benign plexiform neurofibromas and diffuse cutaneous neurofibromas. We investigated the presence and distribution of somatic mitochondrial DNA (mtDNA) mutations in neurofibromas and in nontumor tissue of neurofibromatosis type 1 patients. MtDNA alterations in the entire mitochondrial genome were analyzed by temporal temperature gradient gel electrophoresis followed by DNA sequencing. Somatic mtDNA mutations in tumors were found in 7 of 19 individuals with cutaneous neurofibromas and in 9 of 18 patients with plexiform neurofibromas. A total of 34 somatic mtDNA mutations were found. All mutations were located in the displacement loop region of the mitochondrial genome. Several plexiform neurofibromas from individual patients had multiple homoplasmic mtDNA mutations. In cutaneous neurofibromas, the same mtDNA mutations were always present in tumors from different locations of the same individual. An increase in the proportion of the mutant mtDNA was always found in the neurofibromas when compared with nontumor tissues. The somatic mtDNA mutations were present in the Schwann cells of the analyzed multiple cutaneous neurofibromas of the same individual. The observed dominance of a single mtDNA mutation in multiple cutaneous neurofibromas of individual patients indicates a common tumor cell ancestry and suggests a replicative advantage rather than random segregation for cells carrying these mutated mitochondria.     

Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples

Long RT-PCR (LRP) amplification of RNA templates is sometimes difficult compared to long PCR of DNA templates. Among RNA templates, hepatitis C virus (HCV) represents an excellent example to challenge the potential of LRP technology due to its extensive secondary structures and its difficulty to be readily cultured in vitro. The only source for viral genome amplification is clinical samples in which HCV is usually present at low titers. We have created a comprehensive optimization protocol that allows robust amplification of a 9.1 kb fragment of HCV, followed by efficient cloning into a novel vector. Detailed analyses indicate the lack of potential LRP-mediated recombination and the preservation of viral diversity. Thus, our LRP protocol could be applied for the amplification of other difficult RNA templates and may facilitate RNA virus research such as linked viral mutations and reverse genetics.     

scDPN for High-throughput Single-cell CNV Detection to Uncover Clonal Evolution During HCC Recurrence

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution. Unfortunately, classical DNA amplification-based methods have low throughput and introduce coverage bias during sample preamplification. We developed a single-cell DNA library preparation method without preamplification in nanolitre scale (scDPN) to address these issues. The method achieved a throughput of up to 1800 cells per run for copy number variation (CNV) detection. Also, our approach demonstrated a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy for cell line and tumor tissue evaluation. We used this approach to profile the tumor clones in paired primary and relapsed tumor samples of hepato-cellular carcinoma (HCC). We identified three clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumor containing additional alterations in chro-mosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumor, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome hetero-geneity and evolution.     

Whole genome amplification from plant cell colonies of somatic hybrids using strand displacement amplification

The application of strand displacement amplification (SDA) is demonstrated for whole genome amplification from nanograms to micrograms for DNA isolated from small plant cell colonies. Secondary digest amplified fragment length polymorphism (SD-AFLP) analysis confirmed that the amplified genome is a representative of the entire genome. This approach allows the amplification of DNA isolated from small cell colonies of putative somatic hybrids for rapid molecular confirmation of the hybrid status of fusion products.     

Restriction landmark genome scanning

Restriction landmark genome scanning (RLGS) is a quantitative approach that is uniquely suited for simultaneously assessing the methylation status of thousands of CpG islands. RLGS separates radiolabeled NotI fragments (or other CpG-containing restriction enzyme fragments) in two dimensions and allows distinction of single-copy CpG islands from multicopy CpG-rich sequences. The methylation sensitivity of the endonuclease activity of NotI provides the basis for differential methylation analysis, and NotI sites occur primarily in CpG islands and genes. RLGS has been used to identify novel imprinted genes, novel targets of DNA amplification and methylation in human cancer, and to identify deletion, methylation, and gene amplification in a mouse model of tumorigenesis. Such massively parallel analyses are critical for pattern recognition within and between tumor types, and for estimating the overall influence of CpG island methylation on the cancer cell genome. RLGS is also a useful method for integrating methylation analyses with high-resolution gene copy number analyses.     

Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples

Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a φ29 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.     

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 10⁵ droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.     

Gene amplification in cancer

Gene amplification is a copy number increase of a restricted region of a chromosome arm. It is prevalent in some tumors and is associated with overexpression of the amplified gene(s). Amplified DNA can be organized as extrachromosomal elements, as repeated units at a single locus or scattered throughout the genome. Common chromosomal fragile sites, defects in DNA replication or telomere dysfunction might promote amplification. Some regions of amplification are complex, yet elements of the pattern are reproduced in different tumor types. A genetic basis for amplification is suggested by its relative frequency in some tumor subtypes, and its occurrence in "early" preneoplastic lesions. Clinically, amplification has prognostic and diagnostic usefulness, and is a mechanism of acquired drug resistance.     

Testing the Reproducibility of Multiple Displacement Amplification on Genomes of Clonal Endosymbiont Populations

The multiple displacement amplification method has revolutionized genomic studies of uncultured bacteria, where the extraction of pure DNA in sufficient quantity for next-generation sequencing is challenging. However, the method is problematic in that it amplifies the target DNA unevenly, induces the formation of chimeric reads and also amplifies contaminating DNA. Here, we have tested the reproducibility of the multiple displacement amplification method using serial dilutions of extracted genomic DNA and intact cells from the cultured endosymbiont Bartonella australis. The amplified DNA was sequenced with the Illumina sequencing technology, and the results were compared to sequence data obtained from unamplified DNA in this study as well as from a previously published genome project. We show that artifacts such as the extent of the amplification bias, the percentage of chimeric reads and the relative fraction of contaminating DNA increase dramatically for the smallest amounts of template DNA. The pattern of read coverage was reproducibly obtained for samples with higher amounts of template DNA, suggesting that the bias is non-random and genome-specific. A re-analysis of previously published sequence data obtained after amplification from clonal endosymbiont populations confirmed these predictions. We conclude that many of the artifacts associated with the use of the multiple displacement amplification method can be alleviated or much reduced by using multiple cells as the template for the amplification. These findings should be particularly useful for researchers studying the genomes of endosymbionts and other uncultured bacteria, for which a small clonal population of cells can be isolated.     

Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies

We assessed the whole genome amplification strategy, known as multiple displacement amplification (MDA), for use with the TaqMan genotyping platform for DNA samples derived from two case-control studies nested in the Nurses' Health Study and the Physicians' Health Study. Our objectives were to (1) quantify DNA yield from samples of varying starting concentrations and (2) assess whether MDA products give an accurate representation of the original genomic sequence. Multiple displacement amplification yielded a mean 23000-fold increase in DNA quantity and genotyping results demonstrate 99.95% accuracy across six SNPs from four genes for 352 samples included in this study. These results suggest that MDA will provide a sufficiently robust amplification of limiting samples of genomic DNA that can be used for SNP genotyping in large case-control studies of complex diseases.     

Decontamination of MDA reagents for single cell whole genome amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.     

Comparison of Amplification Methods of Single Trophoblast Cells for Their Subsequent Analysis Using Metaphase Comparative Genomic Hybridization

Single trophoblast cells circulating in the bloodstream of pregnant women are potential objects for noninvasive prenatal diagnosis. Owing to the very low concentration of cells of a fetal nature in the peripheral maternal blood, the choice of the method for whole genome amplification of the genetic material becomes topical. The key point in the use of single cells of a fetal nature for noninvasive prenatal diagnosis is to obtain DNA in an amount and of a quality acceptable for the analysis. In order to select the optimal method for whole genome amplification, a model experiment was conducted. We compared three different methods of whole genome amplification: linker-adaptor polymerase chain reaction (LA-PCR), degenerate oligonucleotide- primed PCR (DOP-PCR), and multiple displacement amplification (MDA). Subsequent analysis of the amplification products was performed by metaphase comparative genomic hybridization in order to evaluate the molecular karyotype of cells of a fetal nature with the known chromosome complement. As a result, an optimal method for whole genome amplification of the genetic material of single cells in a model experiment was determined by linker-adaptor PCR, which showed a more uniform representation of the genome regions compared with the other methods used.     

DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis.

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.