Comparison of seven single cell whole genome amplification commercial kits using targeted sequencing.
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ABSTRACT:
SUBMITTER: Biezuner T
PROVIDER: S-EPMC8387353 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Advances in biochemical technologies have led to a boost in the field of single cell genomics. Observation of the genome at a single cell resolution is currently achieved by pre-amplification using whole genome amplification (WGA) techniques that differ by their biochemical aspects and as a result by biased amplification of the original molecule. Several comparisons between commercially available single cell dedicated WGA kits (scWGA) were performed, however, these comparisons are costly, were only performed on selected scWGA kit and more notably, are limited by the number of analyzed cells, making them limited for reproducibility analysis. We benchmarked an economical assay to compare all commercially available scWGA kits that is based on targeted sequencing of thousands of genomic regions, including highly mutable genomic regions (microsatellites), from a large cohort of human single cells (125 cells in total). Using this approach, we could analyze the genome coverage, the reproducibility of genome coverage and the error rate of each kit. Our experimental design provides an affordable and reliable comparative assay that simulates a real single cell experiment. Results demonstrate the need for a dedicated kit selection depending on the desired single cell assay.
2017-09-24 | E-MTAB-5968 | biostudies-arrayexpress
Project description:Comparison of seven single cell Whole Genome Amplification commercial kits using targeted sequencing
| PRJEB22132 | ENA
Project description:The final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed. The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene). We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95 % limit of detection (LOD95). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n = 13) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n = 6) and a panel of RNA from related human coronaviruses to evaluate assay specificity. PCR efficiency was ≥96 % for all assays and the estimated LOD95 varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene. We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.
| S-EPMC7206434 | biostudies-literature
Project description:BackgroundWhole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly.ResultsThe largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling. Conclusively, the products from the AMPLI1 and MALBAC kits were shown to be most similar to the bulk samples and are therefore recommended for WGA of single cells.DiscussionIn this study four commercial kits for WGA (AMPLI1, MALBAC, Repli-G and PicoPlex) were used to amplify human single cells. The WGA products were exome sequenced together with non-amplified bulk samples from the same source. The resulting data was evaluated in terms of genomic coverage, allelic dropout and SNP calling.
| S-EPMC5313163 | biostudies-literature
Project description:Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.
| S-EPMC5862989 | biostudies-literature
Project description:Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.
| S-EPMC4138190 | biostudies-literature
Project description:Mitochondrial dysfunction has emerged to be associated with a broad spectrum of diseases, and there is an increasing demand for accurate detection of mitochondrial DNA (mtDNA) variants. Whole genome sequencing (WGS) has been the dominant sequencing approach to identify genetic variants in recent decades, but most studies focus on variants on the nuclear genome. Whole genome sequencing is also costly and time consuming. Sequencing specifically targeted for mtDNA is commonly used in the diagnostic settings and has lower costs. However, there is a lack of pairwise comparisons between these two sequencing approaches for calling mtDNA variants on a population basis. In this study, we compared WGS and mtDNA-targeted sequencing (targeted-seq) in analyzing mitochondrial DNA from 1499 participants recruited into the Severe Asthma Research Program (SARP). Our study reveals that targeted-sequencing and WGS have comparable capacity to determine genotypes and to call haplogroups and homoplasmies on mtDNA. However, there exists a large variability in calling heteroplasmies, especially for low-frequency heteroplasmies, which indicates that investigators should be cautious about heteroplasmies acquired from different sequencing methods. Further research is highly desired to improve variant detection methods for mitochondrial DNA.
| S-EPMC8354572 | biostudies-literature
Project description:Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.
| S-EPMC5442535 | biostudies-literature
Project description:Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnosis, prognosis and monitoring treatment of disease. However, a sensitive and specific whole-genome sequencing (WGS) method is required to identify novel genetic variations (i.e., SNVs, CNVs and INDELS) on ccfDNA that can be used as clinical biomarkers. In this article, five WGS methods were compared: ThruPLEX Plasma-seq, QIAseq cfDNA All-in-One, NEXTFLEX Cell Free DNA-seq, Accel-NGS 2 S PCR FREE DNA and Accel-NGS 2 S PLUS DNA. The Accel PCR-free kit did not produce enough material for sequencing. The other kits had significant common number of SNVs, INDELs and CNVs and showed similar results for SNVs and CNVs. The detection of variants and genomic signatures depends more upon the type of plasma sample rather than the WGS method used. Accel detected several variants not observed by the other kits. ThruPLEX seemed to identify more low-abundant SNVs and SNV signatures were similar to signatures observed with the QIAseq kit. Accel and NEXTFLEX had similar CNV and SNV signatures. These results demonstrate the importance of establishing a standardized workflow for identifying non-invasive candidate biomarkers. Moreover, the combination of variants discovered in ccfDNA using WGS has the potential to identify enrichment pathways, while the analysis of signatures could identify new subgroups of patients.
| S-EPMC7148341 | biostudies-literature
Project description:Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.
| S-EPMC5507899 | biostudies-literature