Whole Genome Amplification Methods And Protocols

By Thomas Kroneis

  • Genre : Biology
  • Publisher :
  • ISBN : 978 1493929894
  • Year : 2015
  • Language: English


Preface It is the nature of lab techniques that they steadily improve The development of highresolution analysis such as comparative genome hybridization or the need of multiple analyses from minute amounts of template down to the single-cell level entails improved sample preparation Hence amplification of template DNA is necessary to meet the requirements of state-of-the-art analysis Although not representing an endpoint in analysis equal and unbiased amplification of DNA is a conditio sine qua non for downstream methods and so whole genome amplification WGA methods were developed adapted and optimized in parallel and with regard to the lab techniques that allowed for increasingly powerful analyses Apart from methods longing for a certain amount of starting template WGA is also necessary in case of multiple analyses to be done from low-template or single-cell samples This may not only allow for repeated analyses of one and the same sample but also for combining diverse strategies such as targeted and screening approaches This volume on whole genome amplification is meant to mirror the holistic feature of WGA technology by combining reviews detailed basic methods and advanced sample workflows In the first chapters the interested reader will gain an overview of the development of WGA techniques throughout the recent years Furthermore general considerations on bias in WGA and how to run a single-cell lab are given and possible sample pre-enrichment strategies are introduced The second part focuses on major WGA methods and most important also protocols allowing assessing the WGA product quality Covering most chapters the final part contains advanced protocols that go hand in hand with WGA These chapters address issues such as sample preparation using laser microdissection WGA from partially degraded DNA formalin-fixed paraffin-embedded samples circulating tumor cells or even ancient samples Following the techniques described here will most likely result in successful whole genome amplification even though some of the described methods e g laser microdissection require some attention and improve with practice Thus this volume shall enable the newcomer to get started and yield results within short time and serve old hands as rich source of detailed information and inspiration Graz Austria Thomas Kroneis v Chapter 1 Principles of Whole-Genome Amplification Zbigniew Tadeusz Czyz Stefan Kirsch and Bernhard Polzer Abstract Modern molecular biology relies on large amounts of high-quality genomic DNA However in a number of clinical or biological applications this requirement cannot be met as starting material is either limited e g preimplantation genetic diagnosis PGD or analysis of minimal residual cancer or of insufficient quality e g formalin-fixed paraffin-embedded tissue samples or forensics As a consequence in order to obtain sufficient amounts of material to analyze these demanding samples by state-of-the-art modern molecular assays genomic DNA has to be amplified This chapter summarizes available technologies for whole-genome amplification WGA bridging the last 25 years from the first developments to currently applied methods We will especially elaborate on research application as well as inherent advantages and limitations of various WGA technologies Key words Whole-genome amplification PCR-based amplification Ligation-mediated amplification Multiple displacement amplification 1 Challenges of Analyzing Minimal Quantities of Genomic DNA For most high-throughput assays in molecular biology large amounts of high-quality genomic DNA gDNA are needed as starting material However depending on the source of the sample its inherent characteristics and the spectrum of downstream analyses this requirement simply cannot be met In some applications e g prenatal genetic diagnostics PDG or minimal residual cancer the amount of starting material is extremely limited and often restricted to only one individual cell which corresponds to approximately 7 pg of gDNA 1 Additionally in some instances as in forensics paleobiology or when processing formalin-fixed paraffin-embedded tissue FFPE specimens the sample processing procedures and or storage may diminish the quality and quantity of the available DNA In these cases direct analysis of the sample s gDNA is technically challenging and enables assessment of only limited amount of genetic markers Thomas Kroneis ed Whole Genome Amplification Methods and Protocols Methods in Molecular Biology vol 1347 DOI 10 1007 978-1-4939-2990-0 1 Springer Science Business Media New York 2015 1 2 Zbigniew Tadeusz Czyz et al To enable comprehensive analysis of such demanding samples by modern molecular assays gDNA has to be amplified The amplification procedure has to 1 ensure high genomic coverage that is as much as possible of all 3 109 nucleotides comprising the human genome have to be amplified 2 maintain the inherent sequence composition that is avoid artificial loss of one or even both gene copies maternal and paternal without introducing artificial sequence variation and 3 allow reliable quantification of copy number variation that is all regions of the genome have to be amplified homogeneously During the last decades several methods for whole-genome amplification WGA have been developed Most of them rely on the principle of polymerase chain reaction PCR a powerful technique allowing exponential amplification of the DNA using thermostable DNA polymerases and short oligonucleotide primers 2 In its original form PCR was designed to amplify specific DNA loci with limited amplicon length due to the processivity of the polymerase For example Taq DNA polymerase which is typically used in PCR is amenable to generate amplicons of approximately 1000 base pair in length Thus to amplify whole genomes three basic principles have been applied 1 increasing the amount of priming events 2 reducing the complexity of the genome prior to amplification i e by fragmentation in smaller fragments or 3 utilizing alternative enzymes with higher processivity 2 First Attempts to Amplify Whole Genomes by PCR-Based Technologies The first approach to amplify a genome used non-degenerated primers targeting the most conserved regions of repetitive Alu motifs within the genome 3 This method called interspersed repetitive sequence IRS PCR allows amplification of fragments directly adjacent to Alu elements Although Alu elements are abundantly present within the human genome their distribution within the genome is not uniform 4 which results in a bias towards amplification of regions enriched for Alu sequences 5 Additionally Alu sequences are not frequent for some other species e g mouse decreasing the applicability of the method for single cells in studies involving animal models 5 IRS PCR was primarily used for generation of probe libraries specific for designated regions of the human genome from either mixed DNA sources i e human rodent somatic cell hybrids 6 8 or microdissected human chromosomes 9 One way to assure a more uniform distribution of the priming events across the genome is partial or complete randomization of primer sequences Degenerate oligonucleotide primed DOP PCR is based on the assumption that random primers anneal uniformly across the genome 10 The method utilizes primers Principles of Whole-Genome Amplification 5 enzymatic treatment i e Omniplex GenomePlex technology by Rubicon Genomics 36 37 The method is less dependent on the quality of the starting material as following the fragmentation and fill-in reaction PCR adaptors can be ligated even to partially degraded template e g in FFPE tissue specimens Moreover due to the use of non-degenerated primers this approach ensures constant priming efficiency which results in high DNA yields Nevertheless the priming pattern remains erratic and unreproducible as in PEPand DOP-PCR Thus far LM-PCR-based GenomePlex technology has been used in a variety of applications The method enabled analysis of as little as 5 ng of FFPE-derived DNA material 38 or as few as 2000 microdissected cells by aCGH 39 Moreover GenomePlex technology was successfully applied for aCGH-based analysis of single disseminated cancer cells 40 42 and enriched populations of circulating tumor cells 43 44 and single human blastomeres 45 Here however due to high-level technical noise designated algorithms had to be developed to allow evaluation of single-cell aCGH data sets 41 More recently GenomePlex technology was used for low-coverage high-throughput sequencing of single-cell nuclei sorted by FACS 46 Based on profiles of copy number changes this approach allowed to investigate the population structure and evolution of tumorigenic clones in individual tumor specimens 46 Massive parallel sequencing was also used to sequence a panel of cancer-related genes in single-cell GenomePlex WGA products generated from colorectal cancer circulating tumor cells facilitating discovery of discrepancies in mutation spectrum between primary tumor metastasis and corresponding tumor cells in circulation of the same cancer patients 47 Another fully deterministic WGA technology utilizing the LM-PCR approach is the single-cell comparative genomic hybridization SCOMP published by Klein and colleagues 48 In this approach the fragmented representation of the genome is achieved by the use of restriction endonuclease MseI whose 4-base pairlong restriction motif is distributed with an average spacing of 126 base pair across the human genome based on the hg19 build of the human genome an optimal size for the subsequent PCRbased amplification Still this approach may be less favorable for templates with more infrequent distribution of MseI restriction sites Following the restriction digestion PCR-adaptor sequences are ligated to the MseI representation of the genome assuring a deterministic reproducible priming pattern and high genomic coverage of WGA Furthermore the designated design of the PCR adaptors used in SCOMP minimized the risk of their multimerization and undesirable mispriming within the sampled genome thereby improving the efficiency of the PCR Collectively the unique design of SCOMP makes it particularly advantageous for the downstream assays wherein reproducible coverage and 6 Zbigniew Tadeusz Czyz et al composition of the input material are essential for the analysis i e genotyping and targeted sequence analyses This aspect is particularly important for the diagnostic assays used in the clinic SCOMP was successfully applied to amplify single-cell DNA of disseminated and circulating tumor cells allowing a number of locusspecific analyses i e direct Sanger sequencing- RFLP- and STRbased detection of LOHs 48 50 Importantly comprehensive representation of the single-cell DNA by SCOMP was demonstrated by metaphase CGH allowing detection of copy number alterations CNAs in single tumor cells in numerous studies 48 49 51 54 Moreover SCOMP facilitated successful metaphase CGH-based analysis of single human blastomeres allowing detection of unbalanced translocations and mosaicism within individual embryos 55 More recently products of SCOMP were analyzed using a BAC clone-based 56 and oligonucleotide aCGH platforms 57 58 providing high-quality data and allowing detection of CNAs as small as 53 kb in size Moreover applicability of SCOMP was also demonstrated for dissected FFPE tissue sections providing high-quality results of both metaphase and array-based CGH 59 60 In both of these studies SCOMP outperformed DOP-PCR allowing more accurate and unbiased analysis of copy number changes The unique deterministic design and power of the method in the analysis of clinical material e g disseminated cancer cells and FFPE tissue specimens recently led to commercialization of the principle method as Ampli1 WGA kit Silicon Biosystems SpA Bologna Italy 4 High Processivity by MDA-Based WGA Strand displacement amplification SDA or multiple displacement amplification MDA 61 is based on rolling circle amplification a replication mechanism naturally occurring in the and various other bacteriophages 62 The method was initially adapted to amplify circular DNA templates 63 and later also used for WGA of single cells 12 MDA utilizes enzymes as the highly processive Phi29 or Bst DNA polymerases 12 64 with proofreading activity 65 In principle exonuclease-resistant random hexamers bind to denatured DNA followed by an isothermal amplification 12 66 As a consequence of the strong strand displacement activity of the applied polymerase generated fragments become available for secondary priming events leading to the generation of a network of hyperbranched structures and thus multiple overlapping copies of the starting material High processivity of the Phi29 DNA polymerase results in generation of relatively large amplicons 10 kb 12 facilitating high genomic coverage of single-cell genomes 67 Notably however utilization of MDA leads to considerable sequence representation bias 68 and a tendency to generate Principles of Whole-Genome Amplification 7 chimeric DNA rearrangements in the amplified DNA 69 This results in a significant rate of allelic dropout ADO 70 or preferential amplification PA particularly affecting highly polymorphic sequences 71 These effects are further pronounced in samples with low DNA quantity 64 72 and especially with fragmented template DNA 73 74 This reasoning does not support the application of MDA on clinical samples as e g circulating tumor cells for which fixation and transport logistics may lead to considerable degradation of high-molecular DNA For these types of samples PCR-based WGA approaches were suggested as the better alternative than MDA 75 Despite the mentioned shortcomings MDA technology has been applied in numerous studies on single-cell DNA i e for genotyping of short tandem repeats 76 77 assessment of copy number changes by CGH 5 or aCGH 64 78 79 and more recently whole-exome or whole-genome sequencing 80 83 Furthermore DNA yields after MDA-based amplification are sufficient to allow multiple downstream analysis with the same single-cell sample 84 As MDA-based WGA is an easyto-use and efficient approach to generate large quantities of genomic DNA from small sample sizes it has been commercialized e g Qiagen s REPLI-g product family and GenomiPhi by GE Healthcare and is widely used to generate a long-lasting source of DNA for downstream genetic analyses of small sample sizes 5 WGA Approaches Combining MDA and PCR In an effort to synergize the advantages and negate the disadvantages of both MDA- and PCR-based WGA the company Rubicon Genomics developed PicoPlex the first technology to combine both WGA principles 85 Apart from the patent holder Rubicon Genomics this WGA technology is also vended by the New England Biolabs Inc Single Cell WGA kit BlueGnome SurePLEX and Perkin Elmer EasyAmp In the PicoPlex WGA protocol genomic DNA is initially amplified in an MDA-based process utilizing a set of four non-selfcomplementary primers These so-called self-inert primers are composed of base pair combinations that do not participate in the Watson-Crick base pairing i e A C A G T C and T G Through this intervention formation of primer dimers is precluded which has a strong positive impact on the efficiency of the reaction Selfinert primers are composed of two sections degenerated sequence at the 3 -end responsible for frequent priming in the genome and the fixed sequence at the 5 -end During the initial MDA-based pre-amplification step fixed sequences are incorporated to the end of each amplicon In a second step these molecules are amplified by Principles of Whole-Genome Amplification 9 detection of aneuploidy and single-nucleotide variants In a recent study on CTCs of lung cancer patients single-cell genomes were analyzed by exome and whole-genome sequencing after CellSearch detection and MALBAC 98 Although putative druggable copy number variations and sequence variations could be discovered high allelic dropout at the single-cell level could be observed 98 questioning the reliable use of the method on fixed single CTCs from cancer patients Recently MALBAC has been made commercially available by Yikon Genomics Beijing China 6 Conclusion The increasing number of publications and commercialized molecular methods to analyze genomes of single cells as reviewed in this chapter depicts the increased interest in studies on cellular heterogeneity In the future the link between inherent genomic changes of individual cells with their functional state would certainly improve our understanding on clonal evolution and cellular adaptation One possibility to achieve this goal is the analysis of genome and transcriptome of the same individual cell as demonstrated by the group of Christoph Klein 99 Correlation of results obtained by this approach with the cellular phenotype of individual cells as e g by multicolor immunostaining would provide a basis to 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Author Thomas Kroneis Isbn 978 1493929894 File size 4 6 MB Year 2015 Pages 704 Language English File format PDF Category Biology This volume mirrors the holistic feature of whole genome amplification WGA technology by combining reviews detailed basic methods and advanced sample workflows The first part of the book covers an overview of the development of WGA techniques throughout recent years including general considerations on bias in WGA possible sample pre enrichment strategies and how