Whole-genome multiple displacement amplification from single cells

2006 ◽  
Vol 1 (4) ◽  
pp. 1965-1970 ◽  
Author(s):  
Claudia Spits ◽  
Cédric Le Caignec ◽  
Martine De Rycke ◽  
Lindsey Van Haute ◽  
André Van Steirteghem ◽  
...  
Keyword(s):  
Multiple Displacement Amplification ◽  
Single Cells ◽  
Whole Genome

scholarly journals Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates

2016 ◽  
Vol 113 (30) ◽  
pp. 8484-8489 ◽  
Author(s):  
Kaston Leung ◽  
Anders Klaus ◽  
Bill K. Lin ◽  
Emma Laks ◽  
Justina Biele ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Single Cell ◽  
High Throughput ◽  
Multiple Displacement Amplification ◽  
Single Cells ◽  
Biological Significance ◽  
Phase Cell ◽  
Whole Genome ◽  
Large Numbers ◽  
Diploid Cells

The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10−6 and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.

scholarly journals Quantifying genome DNA during whole-genome amplification via quantitative real-time multiple displacement amplification

RSC Advances ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 4617-4621
Author(s):  
Jing Tu ◽  
Yi Qiao ◽  
Yuhan Luo ◽  
Naiyun Long ◽  
Zuhong Lu
Keyword(s):  
Real Time ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Whole Genome ◽  
Genome Amplification

Monitoring multiple displacement amplification by fluorescence signals.

scholarly journals Development of Microbial Genome-Probing Microarrays using Digital Multiple Displacement Amplification of Uncultivated Microbial Single Cells

2008 ◽  
Vol 42 (22) ◽  
pp. 8614-8614
Author(s):  
Ho-Won Chang ◽  
Youboong Sung ◽  
Kyoung-Ho Kim ◽  
Young-Do Nam ◽  
Seong Woon Roh ◽  
...  
Keyword(s):  
Multiple Displacement Amplification ◽  
Single Cells ◽  
Microbial Genome

scholarly journals MBRS-59. SINGLE-CELL WHOLE-GENOME SEQUENCING DISSECTS INTRA-TUMOURAL GENOMIC HETEROGENEITY AND CLONAL EVOLUTION IN CHILDHOOD MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii408-iii408
Author(s):  
Marina Danilenko ◽  
Masood Zaka ◽  
Claire Keeling ◽  
Stephen Crosier ◽  
Rafiqul Hussain ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Single Cell ◽  
Genome Sequencing ◽  
Copy Number ◽  
Single Cell Analysis ◽  
Mutational Analysis ◽  
Single Cells ◽  
Clonal Evolution ◽  
Whole Genome ◽  
Clinically Significant

Abstract Medulloblastomas harbor clinically-significant intra-tumoral heterogeneity for key biomarkers (e.g. MYC/MYCN, β-catenin). Recent studies have characterized transcriptional heterogeneity at the single-cell level, however the underlying genomic copy number and mutational architecture remains to be resolved. We therefore sought to establish the intra-tumoural genomic heterogeneity of medulloblastoma at single-cell resolution. Copy number patterns were dissected by whole-genome sequencing in 1024 single cells isolated from multiple distinct tumour regions within 16 snap-frozen medulloblastomas, representing the major molecular subgroups (WNT, SHH, Group3, Group4) and genotypes (i.e. MYC amplification, TP53 mutation). Common copy number driver and subclonal events were identified, providing clear evidence of copy number evolution in medulloblastoma development. Moreover, subclonal whole-arm and focal copy number alterations covering important genomic loci (e.g. on chr10 of SHH patients) were detected in single tumour cells, yet undetectable at the bulk-tumor level. Spatial copy number heterogeneity was also common, with differences between clonal and subclonal events detected in distinct regions of individual tumours. Mutational analysis of the cells allowed dissection of spatial and clonal heterogeneity patterns for key medulloblastoma mutations (e.g. CTNNB1, TP53, SMARCA4, PTCH1) within our cohort. Integrated copy number and mutational analysis is underway to establish their inter-relationships and relative contributions to clonal evolution during tumourigenesis. In summary, single-cell analysis has enabled the resolution of common mutational and copy number drivers, alongside sub-clonal events and distinct patterns of clonal and spatial evolution, in medulloblastoma development. We anticipate these findings will provide a critical foundation for future improved biomarker selection, and the development of targeted therapies.

Abstract A38: Optimization of whole-genome amplification for analysis of single cells using next-generation sequencing

2015 ◽  
Author(s):  
Terry J. Amiss ◽  
Frances Tong ◽  
Eileen Snowden ◽  
Richard Kelly ◽  
Rainer Blaesius ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Whole Genome ◽  
Next Generation ◽  
Genome Amplification ◽  
Generation Sequencing

scholarly journals DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells

2019 ◽  
Vol 47 (19) ◽  
pp. e122-e122
Author(s):  
Ramya Viswanathan ◽  
Elsie Cheruba ◽  
Lih Feng Cheow
Keyword(s):  
Dna Methylation ◽  
Restriction Enzyme ◽  
Copy Number ◽  
Dna Analysis ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Whole Genome ◽  
Copy Number Alterations ◽  
Genome Wide

Abstract Genome-wide profiling of copy number alterations and DNA methylation in single cells could enable detailed investigation into the genomic and epigenomic heterogeneity of complex cell populations. However, current methods to do this require complex sample processing and cleanup steps, lack consistency, or are biased in their genomic representation. Here, we describe a novel single-tube enzymatic method, DNA Analysis by Restriction Enzyme (DARE), to perform deterministic whole genome amplification while preserving DNA methylation information. This method was evaluated on low amounts of DNA and single cells, and provides accurate copy number aberration calling and representative DNA methylation measurement across the whole genome. Single-cell DARE is an attractive and scalable approach for concurrent genomic and epigenomic characterization of cells in a heterogeneous population.

scholarly journals SCELLECTOR: ranking amplification bias in single cells using shallow sequencing

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Vivekananda Sarangi ◽  
Alexandre Jourdon ◽  
Taejeong Bae ◽  
Arijit Panda ◽  
Flora Vaccarino ◽  
...  
Keyword(s):  
Single Cell ◽  
Multiple Displacement Amplification ◽  
Single Cells ◽  
Sequencing Data ◽  
High Coverage ◽  
Amplification Bias ◽  
Single Cell Profiling ◽  
High Concordance ◽  
Human Neuronal Cells

Abstract Background The study of mosaic mutation is important since it has been linked to cancer and various disorders. Single cell sequencing has become a powerful tool to study the genome of individual cells for the detection of mosaic mutations. The amount of DNA in a single cell needs to be amplified before sequencing and multiple displacement amplification (MDA) is widely used owing to its low error rate and long fragment length of amplified DNA. However, the phi29 polymerase used in MDA is sensitive to template fragmentation and presence of sites with DNA damage that can lead to biases such as allelic imbalance, uneven coverage and over representation of C to T mutations. It is therefore important to select cells with uniform amplification to decrease false positives and increase sensitivity for mosaic mutation detection. Results We propose a method, Scellector (single cell selector), which uses haplotype information to detect amplification quality in shallow coverage sequencing data. We tested Scellector on single human neuronal cells, obtained in vitro and amplified by MDA. Qualities were estimated from shallow sequencing with coverage as low as 0.3× per cell and then confirmed using 30× deep coverage sequencing. The high concordance between shallow and high coverage data validated the method. Conclusion Scellector can potentially be used to rank amplifications obtained from single cell platforms relying on a MDA-like amplification step, such as Chromium Single Cell profiling solution.

scholarly journals Combined Molecular Genetic and Cytogenetic Analysis from Single Cells after Isothermal Whole-Genome Amplification

2011 ◽  
Vol 57 (7) ◽  
pp. 1032-1041 ◽  
Author(s):  
Thomas Kroneis ◽  
Jochen B Geigl ◽  
Amin El-Heliebi ◽  
Martina Auer ◽  
Peter Ulz ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Molecular Genetic ◽  
Target Cells ◽  
Whole Genome ◽  
Single Cell Level ◽  
Cell Level ◽  
Genome Amplification

BACKGROUND Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

scholarly journals Whole-genome molecular haplotyping of single cells

2011 ◽  
Vol 29 (1) ◽  
pp. 51-57 ◽  
Author(s):  
H Christina Fan ◽  
Jianbin Wang ◽  
Anastasia Potanina ◽  
Stephen R Quake
Keyword(s):  
Single Cells ◽  
Whole Genome

scholarly journals TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Ángel J. Picher ◽  
Bettina Budeus ◽  
Oliver Wafzig ◽  
Carola Krüger ◽  
Sara García-Gómez ◽  
...  
Keyword(s):  
Whole Genome Amplification ◽  
Single Cells ◽  
Whole Genome ◽  
Genome Amplification ◽  
Novel Method
Sign in / Sign up

Export Citation Format

Share Document