Whole-genome Amplification Technique Research Articles

Whole Genome Amplification in Preimplantation Genetic Testing in the Era of Massively Parallel Sequencing.

Successful whole genome amplification (WGA) is a cornerstone of contemporary preimplantation genetic testing (PGT). Choosing the most suitable WGA technique for PGT can be particularly challenging because each WGA technique performs differently in combination with different downstream processing and detection methods. The aim of this review is to provide insight into the performance and drawbacks of DOP-PCR, MDA and MALBAC, as well as the hybrid WGA techniques most widely used in PGT. As the field of PGT is moving towards a wide adaptation of comprehensive massively parallel sequencing (MPS)-based approaches, we especially focus our review on MPS parameters and detection opportunities of WGA-amplified material, i.e., mappability of reads, uniformity of coverage and its influence on copy number variation analysis, and genomic coverage and its influence on single nucleotide variation calling. The ability of MDA-based WGA solutions to better cover the targeted genome and the ability of PCR-based solutions to provide better uniformity of coverage are highlighted. While numerous comprehensive PGT solutions exploiting different WGA types and adjusted bioinformatic pipelines to detect copy number and single nucleotide changes are available, the ones exploiting MDA appear more advantageous. The opportunity to fully analyse the targeted genome is influenced by the MPS parameters themselves rather than the solely chosen WGA.

Recent advances and application in whole‐genome multiple displacement amplification

BackgroundThe extremely small amount of DNA in a cell makes it difficult to study the whole genome of single cells, so whole‐genome amplification (WGA) is necessary to increase the DNA amount and enable downstream analyses. Multiple displacement amplification (MDA) is the most widely used WGA technique.ResultsCompared with amplification methods based on PCR and other methods, MDA renders high‐quality DNA products and better genome coverage by using phi29 DNA polymerase. Moreover, recently developed advanced MDA technologies such as microreactor MDA, emulsion MDA, and micro‐channel MDA have improved amplification uniformity. Additionally, the development of other novel methods such as TruePrime WGA allows for amplification without primers.ConclusionHere, we reviewed a selection of recently developed MDA methods, their advantages over other WGA methods, and improved MDA‐based technologies, followed by a discussion of future perspectives. With the continuous development of MDA and the successive update of detection technologies, MDA will be applied in increasingly more fields and provide a solid foundation for scientific research.

Rapid preimplantation genetic diagnosis of α-thalassemia SEA deletion with blastocyst cell whole genome amplification and short fragment Gap-PCR method

To develop a rapid preimplantation genetic diagnosis method for α-thalassemia SEA deletion based on blastocyst cell whole genome amplification (WGA) combined with short fragment Gap-PCR. Using multiple displacement amplification (MDA) WGA technique, we established a double-fluorescent PCR system of the housekeeping genes GAPDH and β-actin for WGA quality testing, and a genotyping PCR system of mutant and normal short sequences for α-thalassemia SEA deletion. The sensitivity and accuracy of this method for diagnosis of α-thalassemia SEA deletion were evaluated by detecting lymphocyte samples containing different cell numbers from carriers of SEA deletion. The applicability of this method was evaluated by testing of 12 blastocyst biopsy samples. Detection of lymphocyte samples with different cell numbers using the method developed in this study revealed no ADO in 3-cell samples, and the product quantity of WGA became stable for 4-cell samples. Genotyping of the 10 blastocyst biopsy samples with successful WGA showed a genotype of --SEA/αα in 5 samples and αα/αα in the other 5 samples, which were consistent with the verification results. The method developed in this study is a complete testing process for 4-6 blastocyst biopsy cells to allow rapid, accurate, and cost-effective PGD genotyping of α-thalassemia SEA deletion using short fragment gap-PCR.

Improved DOP-PCR (iDOP-PCR): A robust and simple WGA method for efficient amplification of low copy number genomic DNA.

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA).

Isolation and genotyping of circulating tumor cells (CTCs) is gaining an increasing interest by clinical researchers in oncology not only for investigative purposes, but also for concrete application in clinical practice in terms of diagnosis, prognosis and decision treatment with targeted therapies. For the mutational analysis of single CTCs, the most advanced biotechnology methodology currently available includes the combination of whole genome amplification (WGA) followed by next-generation sequencing (NGS). However, the sequence of these molecular techniques is time-consuming and may also favor operator-dependent errors, related to the procedures themselves that, as in the case of the WGA technique, might affect downstream molecular analyses. A preliminary approach of molecular analysis by NGS on a model of CTCs without previous WGA procedural step was performed. We set-up an artificial sample obtained by spiking the SK-MEL-28 melanoma cell line in normal donor peripheral whole blood. Melanoma cells were first enriched using an AutoMACS® (Miltenyi) cell separator and then isolated as single and pooled CTCs by DEPArray™ System (Silicon Biosystems). NGS analysis, using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies) with the Ion Torrent PGM™ system (Life Technologies), was performed on the SK-MEL-28 cell pellet, a single CTC previously processed with WGA and on 1, 2, 4 and 8 recovered CTCs without WGA pre-amplification. NGS directly carried out on CTCs without WGA showed the same mutations identified in SK-MEL-28 cell line pellet, with a considerable efficiency and avoiding the errors induced by the WGA procedure. We identified a cost-effective, time-saving and reliable methodological approach that could improve the analytical accuracy of the liquid biopsy and appears promising in studying CTCs from cancer patients for both research and clinical purposes.

Whole-Genome Haplotyping of Single Sperm of Daphnia pulex (Crustacea, Anomopoda).

Sequencing the entire genome of single sperm cells can provide valuable information of the distribution of meiotic recombination events in eukaryotic genomes. Here, we provide a description of the experimental work flow for isolating single sperm cells from the microcrustacean Daphnia pulex using fluorescence-activated cell sorting. Moreover, we describe the application of a whole-genome amplification technique (i.e., Multiple Annealing and Looping Based Amplification Cycles method) to single sperm of Daphnia to generate enough DNA for library preparation of next-generation sequencing.

Application of Trusight Myeloid Panel on Whole Genome Amplified DNA in Myelodysplastic Syndrome Patients

Background: Whole genome amplification (WGA) has become an invaluable method for working with small amounts of starting DNA and for preserving limited samples of precious stock material. Next-Generation Sequencing (NGS) techniques can benefit from WGA, but due to their high sensitivity, WGA reliability needs to be certified to ensure an unbiased and accurate amplification of whole genomes. Myelodysplastic Syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. We have performed whole exome sequencing (WES) and targeted deep sequencing in tumoral samples from MDS patients. With the aim to determine if Multiple Displacement Amplification-based WGA can be applied to perform NGS in these type of samples and to obtain valuable results, targeted deep sequencing was performed on both fresh-DNA and WGA-DNA from the same patients.Mehtods: Whole bone marrow samples from four MDS patients were included in the study. WGA was performed in tumoral DNA samples with REPLI-g (Qiagen). WES libraries were generated in tumoral-control paired samples using the SureSelect Human Exome Kit 51Mb v4 (Agilent) and sequenced on an Illumina HiSeq2000. Targeted sequencing libraries were prepared for fresh-DNA and WGA-DNA following the manufacturer specifications for TruSight Myeloid Sequencing Panel protocol (Illumina), and then sequenced on one single run on an Illumina MiSeq. WES sequencing data was analyzed using an in-house pipeline, as previously reported. Targeted sequencing data analysis was performed with theMiSeq Reporter Software (Illumina). Filtering was performed in all cases by eliminating sequencing and mapping errors and by discarding intronic or synonym variants, variants located at highly variable regions or with low coverage, as well as know polymorphisms. Additional filtering was performed by visualization on Integrative Genome Viewer Software v.2.3.72.Results: Regarding targeted sequencing, fresh-DNA samples generated 6 million reads (SD = 1.9 million), with 98.5 % (SD = 0.8) of the mapped reads on-target and a mean target coverage of 12148.8 (SD = 3872.9). WGA-DNA samples yielded about 5.2 million reads (SD = 1.5 million), with 98.3 % (SD = 0.4) of the mapped reads on-target and a mean target coverage of 10447.5x (SD = 2946.3). A mean of 77% of total bases displayed a Q score ≥30, which did not differ between fresh and WGA-DNA.Comparison of all filtered variants within the four pairs revealed a high level of discordance between fresh/WGA samples (Figure 1A). A mean of 86% of the detected variants, considering both fresh and WGA-DNA, were detected at a low frequency (<10%). Therefore, a stricter variant filtering was performed, in which all variants detected at a frequency <10% were removed from further analyses.The pairwise comparison across the paired samples showed a total of 48.1% (SD = 49.3) of common variants, 23.2% (SD = 30.1) of variants exclusively detected in fresh-DNA, and 28.7% (SD = 38.4) of variants exclusively detected in WGA-DNA (Figure 1B). Overall, 100% (n=9) of the common variants were also detected by WES. Regarding fresh-DNA specific variants, 63% (5/8) were seen by WES and 37% (3/8) were not. However, these three variants were detected by targeted sequencing at frequencies between 10-12%. This suggests that even a stricter filtering may be necessary when working with WGA-DNA, or that they were not detected by WES because it was performed at a mean coverage of 60x making it difficult to detect low frequency variants. None of the WGA-DNA specific variants were seen by WES. Taking all these factors into account, we used the fresh-DNA specific variants as the gold standard to further calculate the Positive Predictive Value (PPV) and the sensitivity of the WGA-DNA samples, and thus validate the accuracy of WGA technique in the sample preparation. This revealed a sensitivity of 61.7% (SD = 43.3) and a PPV of 53.3% (SD = 54.2).Conclusions: These findings suggest that WGA methods may introduce errors, that can be detected at a low frequency, and that some bias can be expected, explaining why some variants present the gDNA may be lost during the amplification process. Therefore, we believe that applying WGA before library preparation should be restrained to cases with very limited material source and should be followed by a more in-depth and strict bioinformatics analysis and filtering process. [Display omitted] DisclosuresSole:Celgene: Membership on an entity's Board of Directors or advisory committees.

Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells.

BACKGROUNDWhole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive.RESULTSIn respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most.MATERIALS AND METHODSWe analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms.CONCLUSIONWe demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.

Bias in Whole Genome Amplification: Causes and Considerations.

Whole genome amplification (WGA) is a widely used molecular technique that is becoming increasingly necessary in genetic research on a range of sample types including individual cells, fossilized remains and entire ecosystems. Multiple methods of WGA have been developed, each with specific strengths and weaknesses, but with a common defect in that each method distorts the initial template DNA during the course of amplification. The type, extent, and circumstance of the bias vary with the WGA method and particulars of the template DNA. In this review, we endeavor to discuss the types of bias introduced, the susceptibility of common WGA techniques to these bias types, and the interdependence between bias and characteristics of the template DNA. Finally, we attempt to illustrate some of the criteria specific to the analytical platform and research application that should be considered to enable combination of the appropriate WGA method, template DNA, sequencing platform, and intended use for optimal results.

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

Evaluation of circular DNA substrates for whole genome amplification prior to forensic analysis

Forensic biological evidence often contains low quantities of DNA or substantially degraded DNA which makes samples refractory to genotype analysis. One approach that shows promise to overcome the limited quantity of DNA is whole genome amplification (WGA). One WGA technique, termed rolling circle amplification (RCA), involves the amplification of circular DNA fragments and this study evaluates a single-stranded (ss) DNA ligase enzyme for generating circular DNA templates for RCA WGA. Fast, efficient ligation of several sizes of ssDNA templates was achieved. The enzyme also ligated double-stranded (ds) DNA templates, a novel activity not previously reported. Adapter sequences containing optimal terminal nucleotide ends for increased ligation efficiency were designed and ligation of adapters to template DNA was optimized. Increased amplification of DNA templates was observed following WGA; however, no amplification advantage for ssDNA ligase treatment of templates was evident compared to linear templates. A multi-step process to utilize ssDNA ligase prior to WGA was developed and short tandem repeat (STR) analysis of simulated low template (LT) and fragmented DNA was evaluated. The process resulted in the loss of template DNA and failed STR analysis whereas input of linear genomic DNA template directly into WGA prior to STR analysis improved STR genotyping results compared to non-WGA treated samples. Inclusion of an extreme thermostable single-stranded DNA binding protein (SSB) during WGA also increased DNA yields. While STR artifacts such as peak imbalance, drop-in, and dropout persisted, WGA shows potential for successful genetic profiling of LT and fragmented DNA samples. Further research and development is warranted prior to use of WGA in forensic casework.

Whole genome amplification in preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation, improving the chance of conception for patients at high risk of transmitting specific inherited disorders. This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s. Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD, but there are some inevitable shortcomings limiting the scope of genetic diagnosis. Fortunately, different whole genome amplification (WGA) techniques have been developed to overcome these problems. Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed. Moreover, WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis. In this review, we will focus on the currently available WGA techniques and their applications, as well as the new technical trends from WGA products.

Whole genome amplification: Use of advanced isothermal method

Laboratory method for amplifying genomic deoxyribonucleic acid (DNA) samples aiming to generate more amounts and sufficient quantity DNA for subsequent specific analysis is named whole genome amplification (WGA). This method is only way to increase input material from few cells and limited DNA contents. While PCR-based WGA methods have been under continuous development for over a decade, shortcomings of these methods enforced many researchers to switch to the use of non-PCR-based linear amplification techniques. Moreover, application of high fidelity and high possessive DNA polymerases enabled development of an isothermal WGA technique named multiple displacement amplification (MDA). MDA is not based on PCR and doses not require thermal cycling. It should be noted that, while MDA-based techniques proposed aiming to overcome the drawbacks of PCR-based methods however, MDA is still facing some challenges. It seems that PCR-based WGA methods also have some merits. One of the problems which encountered both MDA and PCR-based methods is in the amplification of degraded DNA templates. WGA methods such as T7-based linear amplification of DNA (TLAD), balanced-PCR amplification and restriction and circularization-aided rolling circle amplification (RCA-RCA) have been suggested to aim at amplification of such DNA templates. Keywords: Whole genome amplification, multiple displacement amplification (MDA), non PCR-based methods

Optimized amplification and fluorescent labeling of small cell samples for genomic array‐CGH

Whole genome amplification (WGA) is usually needed in the genetic analysis of samples containing a low number of cells. In genome-wide analysis of DNA copy numbers by array comparative genomic hybridization (array-CGH) it is very important that the genome is evenly represented throughout the amplified product. All currently available WGA techniques are generating some degree of bias. A way to compensate for this is using a reference sample which is similarly amplified, as the introduced amplification bias will be leveled out. Additionally, direct labeling of the amplified DNA is performed to bypass the currently widely applied random primed labeling, which involves an additional amplification of the product and is introducing extra bias. In this article it is shown that equal processing of the test and reference sample is indeed crucial to generate an optimal array-CGH profile of amplified DNA samples. Also presented here is that the labeling method may significantly effect the array-CGH result, it is shown that with direct chemical labeling using platinum derivates (ULS labeling) optimal array-CGH results are obtained. We show that an optimized WGA strategy for both test and reference sample in combination with direct chemical labeling results in a reliable array-CGH profile of samples as low as a 30 cell equivalent.

Whole genome amplification from a single cell: a new era for preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) is a technique used for determining the genetic status of a single cell biopsied from embryos or oocytes. Genetic analysis from a single cell is both rewarding and challenging, especially in PGD. The starting material is very limited and not replaceable, and the diagnosis has to be made in a very short time. Different whole genome amplification (WGA) techniques have been developed to specifically increase the DNA quantities originating from clinical samples with limited DNA contents. In this review, currently available WGA techniques are introduced and, among them, multiple displacement amplification (MDA) is discussed in detail. MDA generates abundant assay-ready DNA to perform broad panels of genetic assays through its ability to rapidly amplify genomes from single cells. The utilization of MDA for single-cell molecular analysis is expanding at a high rate, and MDA is expected to soon become an integral part of PGD.

Aneuploidy Arises at Early Stages of Apc-Driven Intestinal Tumorigenesis and Pinpoints Conserved Chromosomal Loci of Allelic Imbalance between Mouse and Human

Although chromosomal instability characterizes the majority of human colorectal cancers, the contribution of genes such as adenomatous polyposis coli (APC), KRAS, and p53 to this form of genetic instability is still under debate. Here, we have assessed chromosomal imbalances in tumors from mouse models of intestinal cancer, namely Apc(+/1638N), Apc(+/1638N)/KRAS(V12G), and Apc(+/1638N)/Tp53-/-, by array comparative genomic hybridization. All intestinal adenomas from Apc(+/1638N) mice displayed chromosomal alterations, thus confirming the presence of a chromosomal instability defect at early stages of the adenoma-carcinoma sequence. Moreover, loss of the Tp53 tumor suppressor gene, but not KRAS oncogenic activation, results in an increase of gains and losses of whole chromosomes in the Apc-mutant genetic background. Comparative analysis of the overall genomic alterations found in mouse intestinal tumors allowed us to identify a subset of loci syntenic with human chromosomal regions (eg, 1p34-p36, 12q24, 9q34, and 22q) frequently gained or lost in familial adenomas and sporadic colorectal cancers. The latter indicate that, during intestinal tumor development, the genetic mechanisms and the underlying functional defects are conserved across species. Hence, our array comparative genomic hybridization analysis of Apc-mutant intestinal tumors allows the definition of minimal aneuploidy regions conserved between mouse and human and likely to encompass rate-limiting genes for intestinal tumor initiation and progression.

Sensitive Sequencing Method for KRAS Mutation Detection by Pyrosequencing

Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.

Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan

Multiple displacement amplification (MDA) has emerged as a promising new method of whole genome amplification (WGA) with the potential to generate virtually unlimited genome-equivalent DNA from only a small amount of seed DNA. To date, genome-wide high marker density assessments of MDA–DNA have focussed mainly upon suitability for single nucleotide polymorphism (SNP) genotyping applications. Suitability for short tandem repeat (STR) genotyping has not been investigated in great detail, despite their inherent instability during DNA replication, and the obvious challenge that this presents to WGA techniques. Here, we aimed to assess the applicability of MDA in STR genotyping by conducting a genome-wide scan of 768 STR markers for MDAs of 15 high quality genomic DNAs. We found that MDA genotyping call and accuracy rates were only marginally lower than for genomic DNA. Pooling of three replicate MDAs resulted in a small increase in both call rate and genotyping accuracy. We identified 34 STRs (4.4% of total markers) of which five essentially failed with MDA samples, and 29 of which showed elevated genotyping failures/discrepancies in the MDAs. We emphasise the importance of DNA and MDA quality checks, and the use of appropriate controls to identify problematic STR markers.