Accurate identification of single-nucleotide variants in whole-genome-amplified single cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Dong X
PROVIDER: S-EPMC5408311 | biostudies-literature | 2017 May
REPOSITORIES: biostudies-literature
Publications
Accurate identification of single-nucleotide variants in whole-genome-amplified single cells.
Dong Xiao X Zhang Lei L Milholland Brandon B Lee Moonsook M Maslov Alexander Y AY Wang Tao T Vijg Jan J
Nature methods 20170320 5
Mutation analysis in single-cell genomes is prone to artifacts associated with cell lysis and whole-genome amplification. Here we addressed these issues by developing single-cell multiple displacement amplification (SCMDA) and a general-purpose single-cell-variant caller, SCcaller (https://github.com/biosinodx/SCcaller/). By comparing SCMDA-amplified single cells with unamplified clones from the same population, we validated the procedure as a firm foundation for standardized somatic-mutation an ...[more]
Similar Datasets
Project description:BackgroundTarget enrichment and resequencing is a widely used approach for identification of cancer genes and genetic variants associated with diseases. Although cost effective compared to whole genome sequencing, analysis of many samples constitutes a significant cost, which could be reduced by pooling samples before capture. Another limitation to the number of cancer samples that can be analyzed is often the amount of available tumor DNA. We evaluated the performance of whole genome amplified DNA and the power to detect subclonal somatic single nucleotide variants in non-indexed pools of cancer samples using the HaloPlex technology for target enrichment and next generation sequencing.ResultsWe captured a set of 1528 putative somatic single nucleotide variants and germline SNPs, which were identified by whole genome sequencing, with the HaloPlex technology and sequenced to a depth of 792-1752. We found that the allele fractions of the analyzed variants are well preserved during whole genome amplification and that capture specificity or variant calling is not affected. We detected a large majority of the known single nucleotide variants present uniquely in one sample with allele fractions as low as 0.1 in non-indexed pools of up to ten samples. We also identified and experimentally validated six novel variants in the samples included in the pools.ConclusionOur work demonstrates that whole genome amplified DNA can be used for target enrichment equally well as genomic DNA and that accurate variant detection is possible in non-indexed pools of cancer samples. These findings show that analysis of a large number of samples is feasible at low cost, even when only small amounts of DNA is available, and thereby significantly increases the chances of indentifying recurrent mutations in cancer samples.
| S-EPMC4046713 | biostudies-literature
Project description:BackgroundLarge-scale human sequencing projects have described around a hundred-million single nucleotide variants (SNVs). These studies have predominately involved individuals with European ancestry despite the fact that genetic diversity is expected to be highest in Africa where Homo sapiens evolved and has maintained a large population for the longest time. The African Genome Variation Project examined several African populations but these were all located south of the Sahara. Morocco is on the northwest coast of Africa and mostly lies north of the Sahara, which makes it very attractive for studying genetic diversity. The ancestry of present-day Moroccans is unknown and may be substantially different from Africans found South of the Sahara desert, Recent genomic data of Taforalt individuals in Eastern Morocco revealed 15,000-year-old modern humans and suggested that North African individuals may be genetically distinct from previously studied African populations.ResultsWe present SNVs discovered by whole genome sequencing (WGS) of three Moroccans. From a total of 5.9 million SNVs detected, over 200,000 were not identified by 1000G and were not in the extensive gnomAD database. We summarise the SNVs by genomic position, type of sequence gene context and effect on proteins encoded by the sequence. Analysis of the overall genomic information of the Moroccan individuals to individuals from 1000G supports the Moroccan population being distinct from both sub-Saharan African and European populations.ConclusionsWe conclude that Moroccan samples are genetically distinct and lie in the middle of the previously observed cline between populations of European and African ancestry. WGS of Moroccan individuals can identify a large number of novel SNVs and aid in functional characterisation of the genome.
| S-EPMC7507649 | biostudies-literature
Project description:Current microarray technology allows researchers to genotype a large number of SNPs with relatively small amounts of DNA. Nevertheless, researchers and clinicians still frequently face the problem of acquiring enough high-quality DNA for analysis. Whole-genome amplification (WGA) methods offer a solution for this problem, and earlier studies have shown that WGA samples perform reasonably well in small-scale genetic analyses (e.g. Affymetrix 10K array). To determine the performance of WGA products on a large-scale genotyping array, we compared the Affymetrix 250K array genotyping results of genomic DNA and their WGA products from four individuals. Our results indicate that WGA product performs well on the 250K array compared to genomic DNA, especially when using the BRLMM calling algorithm. WGA samples have high call rates (97.5% on average, compared to 99.4% for genomic DNA) and excellent concordance rates with their corresponding genomic DNA samples (98.7% on average). In addition, no apparent systematic genomic amplification bias can be detected. This study demonstrates that, although there is a slight decrease in the total call rates, WGA methods provide a reliable approach for increasing the amount of DNA samples for use with a common SNP genotyping array.
| S-EPMC2659594 | biostudies-literature
Project description:Massive parallel sequencing (MPS) can accurately quantify mitochondrial DNA (mtDNA) single nucleotide variants (SNVs), but no MPS methods are currently validated to simultaneously and accurately establish the breakpoints and frequency of large deletions at low heteroplasmic loads. Here we present the thorough validation of an MPS protocol to quantify the load of very low frequency, large mtDNA deletions in bulk DNA and single cells, along with SNV calling by standard methods. We used a set of well-characterized DNA samples, DNA mixes and single cells to thoroughly control the study. We developed a custom script for the detection of mtDNA rearrangements that proved to be more accurate in detecting and quantifying deletions than pre-existing tools. We also show that PCR conditions and primersets must be carefully chosen to avoid biases in the retrieved variants and an increase in background noise, and established a lower detection limit of 0.5% heteroplasmic load for large deletions, and 1.5 and 2% for SNVs, for bulk DNA and single cells, respectively. Finally, the analysis of different single cells provided novel insights into mtDNA cellular mosaicism.
| S-EPMC5643964 | biostudies-literature
Project description:BACKGROUND: Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA). We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS) to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS) workflow. RESULTS: The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean) of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models. CONCLUSION: Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.
| S-EPMC3534403 | biostudies-literature
Project description:In large-scale genome-wide association studies based on high-density single nucleotide polymorphism (SNP) genotyping array, the quantity and quality of available genomic DNA (gDNA) is a practical problem. We examined the feasibility of using the Multiple Displacement Amplification (MDA) method of whole-genome amplification (WGA) for such a platform. The Affymetrix Early Access Mendel Nsp 250K GeneChip was used for genotyping 224,940 SNPs per sample for 28 DNA samples. We compared the call concordance using 14 gDNA samples and their corresponding 14 WGA samples. The overall mean genotype call rates in gDNA and the corresponding WGA samples were comparable at 97.07% [95% confidence interval (CI), 96.17-97.97] versus 97.77% (95% CI, 97.26-98.28; P = 0.154), respectively. Reproducibility of the platform, calculated as concordance in duplicate samples, was 99.45%. Overall genotypes for 97.74% (95% CI, 97.03-98.44) of SNPs were concordant between gDNA and WGA samples. When the analysis was restricted to well-performing SNPs (successful genotyping in gDNA and WGA in >90% of samples), 99.11% (95% CI, 98.80-99.42) of the SNPs, on average, were concordant, and overall a SNP showed a discordant call in 0.92% (95% CI, 0.90-0.94) of paired samples. In a pair of gDNA and WGA DNA, similar concordance was reproducible on Illumina's Infinium 610 Quad platform as well. Although copy number analysis revealed a total of seven small telomeric regions in six chromosomes with loss of copy number, the estimated genome representation was 99.29%. In conclusion, our study confirms that high-density oligonucleotide array-based genotyping can yield reproducible data and MDA-WGA DNA products can be effectively used for genome-wide SNP genotyping analysis.
| S-EPMC2871542 | biostudies-literature
Project description:Single-cell genome sequencing provides high-resolution details of the clonal genomic modifications that occur during cancer initiation, progression, and ongoing evolution as patients undergo treatment. One limitation of current single-cell sequencing strategies is a suboptimal capacity to detect all classes of single-nucleotide and structural variants in the same cells.Here we present a new approach for determining comprehensive variant profiles of single cells using a microfluidic amplicon-based strategy to detect structural variant breakpoint sequences instead of using relative read depth to infer copy number changes. This method can reconstruct the clonal architecture and mutational history of a malignancy using all classes and sizes of somatic variants, providing more complete details of the temporal changes in mutational classes and processes that led to the development of a malignant neoplasm. Using this approach, we interrogated cells from a patient with leukemia, determining that processes producing structural variation preceded single nucleotide changes in the development of that malignancy.All classes and sizes of genomic variants can be efficiently detected in single cancer cells using our new method, enabling the ordering of distinct classes of mutations during tumor evolution.
| S-EPMC5702214 | biostudies-literature
Project description:Cattle have been essential for the development of human civilization since their first domestication few thousand years ago. Since then, they have spread across vast geographic areas following human activities. Throughout generations, the cattle genome has been shaped with detectable signals induced by various evolutionary processes, such as natural and human selection processes and demographic events. Identifying such signals, called selection signatures, is one of the primary goals of population genetics. Previous studies used various selection signature methods and normalized the outputs score using specific windows, in kbp or based on the number of SNPs, to identify the candidate regions. The recent method of iSAFE claimed for high accuracy in pinpointing the candidate SNPs. In this study, we analyzed whole-genome resequencing (WGS) data of ten individuals from Austrian Fleckvieh (Bos taurus) and fifty individuals from 14 Chinese indigenous breeds (Bos taurus, Bos taurus indicus, and admixed). Individual WGS reads were aligned to the cattle reference genome of ARS. UCD1.2 and subsequently undergone single nucleotide variants (SNVs) calling pipeline using GATK. Using these SNVs, we examined the population structure using principal component and admixture analysis. Then we refined selection signature candidates using the iSAFE program and compared it with the classical iHS approach. Additionally, we run Fst population differentiation from these two cattle groups. We found gradual changes of taurine in north China to admixed and indicine to the south. Based on the population structure and the number of individuals, we grouped samples to Fleckvieh, three Chinese taurines (Kazakh, Mongolian, Yanbian), admixed individuals (CHBI_Med), indicine individuals (CHBI_Low), and a combination of admixed and indicine (CHBI) for performing iSAFE and iHS tests. There were more significant SNVs identified using iSAFE than the iHS for the candidate of positive selection and more detectable signals in taurine than in indicine individuals. However, combining admixed and indicine individuals decreased the iSAFE signals. From both within-population tests, significant SNVs are linked to the olfactory receptors, production, reproduction, and temperament traits in taurine cattle, while heat and parasites tolerance in the admixed individuals. Fst test suggests similar patterns of population differentiation between Fleckvieh and three Chinese taurine breeds against CHBI. Nevertheless, there are genes shared only among the Chinese taurine, such as PAX5, affecting coat color, which might drive the differences between these yellowish coated breeds, and those in the greater Far East region.
| S-EPMC9552183 | biostudies-literature
Project description:DNA-replication is a key process in life and can lead to disease when disturbed. Cell-type specific early and late replication domains have been discovered throughout genomes by analysis of DNA from populations of cells. However, cell to cell differences and the association of these differences with other cellular processes remain largely elusive. Here we demonstrate for the first time that consecutive domains of early and late DNA-replication can be detected in single S-phase cells using array comparative genomic hybridization, providing proof-of-concept for a novel tool to investigate DNA-replication genome wide at the single-cell level. Furthermore, methods to profile the genome of a single cell for DNA-copy number aberrations are revolutionizing both basic genome research and clinical genetic diagnosis. It is thus important to apprehend not only technical but also biological reasons for false positive copy number detection. None of the current single-cell copy number calling methods distinguishes between a cell in G1-, S- or G2/M-phase of the cell cycle and mostly use cells isolated randomly from populations. We demonstrate that the oscillating pattern between early and late replicating loci instigates significantly more false-positive DNA copy-number calls in a diploid cell in S-phase cells than in G1- or G2/M-phase, depending on the specific aCGH-signal normalization method used. We propose a work-flow to detect single cells in S-phase and to correct for DNA-replication bias before copy number profiling. The genome of 2 S-phase, 1 M-phase and 1 G-phase single cells were amplified using the Sureplex amplification system. These test samples were hybridized comparatively to commercial male reference DNA,
2013-01-08 | E-GEOD-38759 | biostudies-arrayexpress
Project description:Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.
| S-EPMC275486 | biostudies-literature