A pseudolikelihood approach for simultaneous analysis of array comparative genomic hybridizations

DNA sequence copy number has been shown to be associated with cancer development and progression. Array-based comparative genomic hybridization (aCGH) is a recent development that seeks to identify the copy number ratio at large numbers of markers across the genome. Due to experimental and biological variations across chromosomes and hybridizations, current methods are limited to analyses of single chromosomes. We propose a more powerful approach that borrows strength across chromosomes and hybridizations. We assume a Gaussian mixture model, with a hidden Markov dependence structure and with random effects to allow for intertumoral variation, as well as intratumoral clonal variation. For ease of computation, we base estimation on a pseudolikelihood function. The method produces quantitative assessments of the likelihood of genetic alterations at each clone, along with a graphical display for simple visual interpretation. We assess the characteristics of the method through simulation studies and analysis of a brain tumor aCGH data set. We show that the pseudolikelihood approach is superior to existing methods both in detecting small regions of copy number alteration and in accurately classifying regions of change when intratumoral clonal variation is present. Software for this approach is available at http://www.biostat.harvard.edu/ approximately betensky/papers.html.     

Integration Pattern of Human JC Virus Sequences in Two Clones of a Cell Line Established from a JC Virus-Induced Hamster Brain Tumor

The physical state of the JC virus (JCV) genome was studied in two clonal cell lines (clones 2 and 7) derived from a tissue culture cell line (HJC-15) established from a hamster brain tumor induced by JCV. Saturation-hybridization and reassociation kinetic analyses, using in vitro (32)P-labeled JCV DNA, indicated that clone 7 and 2 cells contain 9 to 10 and 4 to 5 copies per cell, respectively, of all or most of the viral genome. Both cell DNAs were analyzed by using the Southern blotting procedure with three restriction endonucleases: XhoI, which does not cleave JCV DNA; EcoRI, which cleaves once; and HindIII, which cleaves three times. With each DNA, a variety of JCV-specific DNA fragments were detected. The following conclusions are possible: (i) JCV DNA is integrated into cell DNA in both clonal lines; (ii) both clonal lines contain multiple copies of the viral genome integrated in a tandem head-to-tail orientation; (iii) neither clonal line contains detectable free-form I, II, or III JCV DNA; (iv) each clonal line contains multiple independent sites of JCV DNA integration; and (v) most or all of the sites of integration on the cellular or the viral genome, or both, are different in clone 7 DNA than in clone 2 DNA. Thus, although both clone 7 and clone 2 cells were established from the HJC-15 tumor cell line, they differ in the copy number and integration pattern of JCV DNA.     

Neuroblastoma epigenetics: From candidate gene approaches to genome-wide screenings

Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system cells. Although recently new insights into genes involved in NB have emerged, the molecular basis of neuroblastoma development and progression still remains poorly understood. The best-characterized genetic alterations include amplification of the proto-oncogene MYCN, ALK activating mutations or amplification, gain of chromosome arm 17q and losses of 1p, 3p, and 11q. Epigenetic alterations have been described as well: caspase-8 (CASP8) and RAS-association domain family 1 isoform A (RASSF1A) DNA-methylation are important events for the development and progression of neuroblastoma. In total, there are about 75 genes described as epigenetically affected in NB cell lines and/or NB primary samples. These epigenetic alterations were either found using a candidate gene approach or based on the analysis of genome-wide screening techniques. This review gives an extensive overview of all epigenetic changes described in NB as of today, with a main focus on both prognostic use and the potential of genome-wide techniques to find epigenetic prognostic biomarkers in NB. We summarize the key findings so far and the state-of-the-art of the upcoming methods at a unique time frame in the transition towards combined genome wide chromatin immune-precipitation (ChIP) and DNA sequencing techniques.     

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.     

Absolute quantification of somatic DNA alterations in human cancer

We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.     

High‐throughput genotyping of Anopheles mosquitoes using intact legs by Agena Biosciences iPLEX

Recent developments in genotyping technologies coupled with the growing desire to characterize genome variation in Anopheles populations open the opportunity to develop more effective genotyping strategies for high‐throughput screening. A major bottleneck of this goal is nucleic acid extraction. Here, we examined the feasibility of using intact portions of a mosquito's leg as sources of template DNA for whole‐genome amplification (WGA) by primer‐extension preamplification. We used the Agena Biosciences MassARRAY^® platform (formerly Sequenom) to genotype 78 SNPs for 265 WGA leg samples. We performed nucleic acid extraction on 36 mosquito carcasses and compared the genotype call concordance with their corresponding legs and observed full concordance. Using three legs instead of one improved genotyping success rates (96% vs. 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, thereby eliminating the need to extract nucleic acid. This approach is straightforward and sensitive and allows both species determination and genotyping of Anopheles mosquitoes to be performed in a high‐throughput manner. Our protocol also leaves the mosquito body intact facilitating other experimental analysis to be undertaken on the same sample. Based on our findings, this method would also be suitable for use with other insect species.     

Improved noninvasive genotyping method: application to brown bear (Ursus arctos) faeces

We redesigned new microsatellite primers and one sex‐specific primer for amplification of faecal DNA from brown bears (Ursus arctos). We also combined a semi‐nested polymerase chain reaction (PCR) with a newly developed multiplex preamplification method in order to increase the quality of the amplified DNA fragments. In comparison with a conventional PCR approach, the genotyping error rate was substantially reduced and the amplification rate was increased. This new approach could be transposed to other species where conventional PCR methods experience low success due to limited DNA concentration and/or quality.     

改良的PEP方法在无创性产前基因诊断中的应用

应用显微操作技术获取孕妇外周血中的单个有核红细胞,改良的PEP方法扩增单个有核红细胞的全基因组DNA;在此基础上,应用荧光标记聚合酶链反应扩增9个微卫星片段,进行基因型分析判定单个有核红细胞来源。综合性别和DMD基因内的数个STR位点连锁分析进行DMD基因诊断,应用PCR-STR连锁分析进行PKU基因诊断。结果显示,对10例DMD高危胎儿中的6例成功地进行了无创性产前基因诊断。同时对1例PKU也成功地进行了无创性产前基因诊断。改良的PEP方法扩增单个细胞的全基因组可以满足基因诊断的要求,是无创性产前基因诊断中一种极有价值的全基因组扩增的方法。 Abstract：We investigated the feasibility of using improved primer extension preamplificat ion method to diagnose DMD and PKU. The fetal nucleated red blood cells from the peripheral blood of pregnant women were detected and individually retrieved into glass capillary pipettes using a micromanipulator under microscopic observation. The whole genome of a single cell was amplified by improved primer extension preamplification (PEP).Genotypes were analyzed by amplifying the 9 STR fragments using fluorescence?PCR technique and NRBC's(nucleated red blood cell) origin w as determined.We diagnosed DMD prenatally using sex determination and linkage an alysis of several STR sites of dystrophin,and we diagnosed PKU prenatally using PCR?STR linkage analysis.6 of 10 potential DMD patients were diagnosed,includin g 1 male fetal patient,1 potential PKU patient was also diagnosed.The improved P EP method is a very valuable method of amplifying the whole genome of single cel ls,and the products of amplification are enough to the requirements of DNA in no n-invasive prenatal diagnosis.     

不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用

单细胞全基因组扩增(whole genome amplification, WGA)是指在单细胞水平对全基因组进行扩增的新技术,其原理是将分离的单个细胞的微量全基因组DNA进行扩增,获得高覆盖率的完整的基因组后进行高通量测序,用于揭示细胞异质性。目前,WGA方法主要包括引物延伸预扩增(primer extension preamplification PCR, PEP-PCR)、简并寡核苷酸引物PCR (degenerate oligonucleotide primed PCR, DOP-PCR)、多重置换扩增(multiple displacement amplification, MDA)、多次退火环状循环扩增(multiple annealing and looping-based amplification cycles, MALBAC)等。本文对不同的单细胞WGA方法的原理及应用情况分别进行了阐述,并对其扩增效率进行评价和比较,包括基因组覆盖度、均一性、重现性、SNV (single-nucleotide variants)和CNV (copy number variants)检测力等。综合对比不同单细胞WGA方法后发现,MALBAC的扩增均一性最高、等位基因脱扣率最低、重现性最好,且对于CNV和SNV的检测效果最好。本文还阐述了MALBAC技术在人类单精子减数重组、非整倍体分析以及人类卵细胞基因组研究中的应用。     

Detection of DNA abnormalities by arbitrarily primed PCR fingerprinting: amplification of the MDM2 gene in a mediastinum fibrosarcoma.

Arbitrarily primed PCR (AP-PCR) fingerprinting method is easy and useful for analysis of genetic alterations in anonymous chromosomal regions. We applied this technology to analysis of DNA from human primary cancers and found amplification of a DNA fragment in a mediastinum fibrosarcoma. PCR-based analysis of radiation hybrid panels following cloning and nucleotide sequence determination of the fragment revealed that it was derived from a region of chromosome 12q13-q15. In this region, the MDM2 and IFNG genes were noted as known genes that could be involved in human carcinogenesis. Southern blot analysis of genomic DNA of the tumor revealed the amplification of the MDM2 gene together with the fragment locus, but not the IFNG gene. Our results demonstrated that detection of DNA alterations by AP-PCR fingerprinting without any previous knowledge of the genes and subsequent analysis of radiation hybrid panels could lead to easy identification of candidates for genes involved in carcinogenesis.     

Environmental whole-genome amplification to access microbial populations in contaminated sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173.     

Development of a low bias method for characterizing viral populations using next generation sequencing technology

Background

With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies.

Methodology/Principal Findings

We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient''s infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity.

Significance

This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure.     

Whole genome amplification of sodium bisulfite-treated DNA allows the accurate estimate of methylated cytosine density in limited DNA resources

Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however is that it results in considerable DNA degradation, and large quantities of genomic DNA material are needed if numerous genomic regions are to be profiled. In this study, we examined whether whole genome amplification (WGA) techniques can be applied to sodium bisulfite-treated DNA and whether WGA would bias DNA methylation results. Sodium bisulfite-treated DNA was amplified using a standard WGA method: optimized primer-extension preamplification (PEP) with degenerate primers. Following the PCR of bisulfite-treated DNA, the DNA methylation profiles of specific DNA fragments were assessed using three approaches: (i) direct sequencing of the overall product; (ii) the sequencing of cloned PCR products; and (iii) methylation-sensitive single nucleotide primer extension (MS-SNuPE)--and compared with those obtained from bisulfite-treated DNA not subjected to WGA. Our data indicates that the DNA methylation profiles obtained from WGA of sodium bisulfite-treated DNA are consistent with those obtained from non-WGA DNA. The average difference in methylation percentage calculated from the two sets of template using MS-SNuPE was 4%. If our results are replicated on other genomic loci, WGA may become a useful technique in DNA methylation studies.     

Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA

The ability to utilize formalin-fixed, paraffin-embedded (FFPE) archival specimens reliably for high-resolution molecular genetic analysis would be of immense practical application in the study of human disease. We have evaluated the ability of the GenomePlex whole genome amplification (WGA) kit to amplify frozen and FFPE tissue for use in array CGH (aCGH). GenomePlex gave highly representative data compared with unamplified controls both from frozen material (Pearson's R(2) = 0.898) and from FFPE (R(2) = 0.883). Artifactual amplification observed using DOP-PCR at chromosomes 1p, 3, 13q, and 16p was not seen with GenomePlex. Highly reproducible aCGH profiles were obtained using as little as 5 ng starting material from FFPE (R(2) = 0.918). This WGA method should readily lend itself to the determination of DNA copy number alterations from small fresh-frozen and FFPE clinical tumor specimens, although some care must be taken to optimize the DNA extraction procedure.     

Phenomenon of evolution of clonal chromosomal abnormalities in childhood acute myeloid leukemia

An analysis of chromosomal abnormalities in bone-marrow cells was performed in 116 children with diagnoses of acute myeloid leukemia (AML). The frequency of the evolution of clonal chromosomal abnormalities in AML constituted 42.3%. Quantitative abnormalities of chromosomes 8, 9, and 21, as well as the secondary structural abnormalities in the chromosomal regions 12p12, 9p22, 9q22, 9q34, 11q14–23, and 6q2, were the most abundant. Quantitative abnormalities were registered in 26.7% cases. The basic mechanism of evolution of the leukemic clone contained trisomy, deletions, and monosomy. The frequency of evolution was seven times higher in the age group of up to 2 years and twofold higher in the age group of up to 5 years. The high frequency of evolution at t(15;17)(q22;q22) was established, while its absence was revealed at inv(16)(p13q22). Patients with clonal evolution were characterized by the increased frequency of relapses and earlier death before reaching remission, which might be explained by the severe initial state of those patients. The conception of abnormalities in the evolution of the clone was proposed to occur at certain stages as follows: (1) appearance of balanced rearrangements; (2) trisomy occurrence; (3) loss of chromosomal material. The occurrence of an unbalanced genome during evolution possesses advantages in the clonal proliferate activity and may be related to its response to chemotherapy. An identity in abnormal chromosomal structure was revealed as a result of the comparison of karyotypes during diagnostics and during relapse, which could be evidence of the initial induction of some types of evolution of chromosomal abnormalities in leukemic cells in AML children by the chemical agents.     

Genetic alterations in brain tumors identified by RAPD analysis

We report the utility of random amplified polymorphic DNA (RAPD) analysis for identifying subtle genomic alterations in meningiomas and gliomas by comparing the DNA band profile of tumor vis-à-vis its constitutional counterpart. Twenty out of the 29 decanucleotide GC-rich random primers utilized for the RAPD analysis of meningiomas revealed alteration(s) in the tumor genome. In gliomas, changes were detected by 16 of the 18 primers. While all the seven meningioma samples exihibited alterations in tumor DNA, changes were evident in 21 of the 24 glioma cases. These alterations in tumor DNA included the loss of a normal band, appearance of a new band and amplification of a pre-existing band. Many primers detected more than one alterations in a given tumor. Our approach, which covers the range from 0.4 to 2 kb, besides detecting a significant number of changes in a spectrum of brain tumors, complements existing DNA fingerprinting methods, such as microsatellite mapping (less than 0.4 kb) and Southern blotting (over 2 kb), for detecting genetic alterations in tumors.     

Single-cell copy number variation detection

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data.     

Hormone resistance and neuroendocrine differentiation due to accumulation of genetic lesions during clonal evolution of prostate cancer

Progression of malignant tumors is largely due to clonal evolution of the primary tumor, clones acquiring different sets of molecular genetic lesions. Lesions can confer a selective advantage in proliferation rate or metastasis on the tumor cell population, especially if developing resistance to anticancer therapy. Prostate cancer (PCa) provides an illustrative example of clinically significant clonal evolution. The review considers the genetic alterations that occur in primary PCa and the mechanism whereby hormone-refractory PCa develops on hormone therapy, including mutations and alternative splicing of the androgen receptor gene (AR) and intratumoral androgen synthesis. Certain molecular genetic lesions determine resistance to new generation inhibitors (AR mutations that block the antagonist effect or allow other hormones to activate the receptor) or lead to neuroendocrine differentiation (repression of the AR signaling pathway, TP53 mutations, and amplification of the AURKA or MYCN oncogene). Multistep therapy based on the data about somatic mutations associated with progression and metastasis of the primary tumor can be expected to significantly improve the survival of patients with advanced PCa in the nearest future.     

Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA

Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.     

Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis.

Rapidly progressive T-cell lymphomas were observed in 3 of 10 rhesus monkeys several months after autologous transplantation of enriched bone marrow stem cells that had been transduced with a retroviral vector preparation containing replication-competent virus (R. E. Donahue, S. W. Kessler, D. Bodice, K. McDonagh, C. Dunbar, S. Goodman, B. Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis, J. Exp. Med. 176:1124-1135, 1992). The animals with lymphoma appeared to be tolerant to retroviral antigens in that their sera lacked antibodies reactive with viral proteins and contained 10(4) to 10(5) infectious virus particles per ml. By molecular cloning and DNA sequencing, we have now demonstrated that the serum from one of the monkeys contained a replication-competent retrovirus that arose by recombination between vector and packaging encoding sequences (vector/helper [V/H] recombinant) in the producer clone used for transduction of bone marrow stem cells. Southern blot analysis demonstrated 14 or 25 copies of this genome per cell where present in two animals. The genome of a second replication-competent virus was also recovered by molecular cloning; it arose by recombination involving the genome of the V/H recombinant and endogenous murine retroviral genomes in the producer clone. Twelve copies of this amphotropic virus/mink cell focus-forming virus genome were present in tumor DNA of one animal, but it was not found in tumor DNA of the other two animals with lymphoma. Southern blot analysis of DNA from various tissues demonstrated common insertion site bands in several samples of tumor DNA from one animal, suggesting clonal origin of the lymphoma. Our data are most consistent with a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis of critical growth control genes, leading to cell transformation and clonal tumor evolution.