scholarly journals HiVA: an integrative wet- and dry-lab platform for haplotype and copy number analysis of single-cell genomes

2019 ◽  
Author(s):  
Masoud Zamani Esteki ◽  
Amin Ardeshirdavani ◽  
Daniel Alcaide ◽  
Heleen Masset ◽  
Jia Ding ◽  
...  
Keyword(s):  
Single Cell ◽  
Genetic Variants ◽  
Copy Number ◽  
Web Application ◽  
Single Cells ◽  
Genomic Data ◽  
Comprehensive Analysis ◽  
Whole Genome ◽  
Copy Number Analysis ◽  
Genome Wide

Haplotyping is imperative for comprehensive analysis of genomes, imputation of genetic variants and interpretation of error-prone single-cell genomic data. Here we present a novel sequencing-based approach for whole-genome SNP typing of single cells, and determine genome-wide haplotypes, the copy number of those haplotypes as well as the parental and segregational origin of chromosomal aberrations from sequencing- and array-based SNP landscapes of single cells. The analytical workflow is made available as an interactive web application HiVA (https://hiva.esat.kuleuven.be).

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.

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.

Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing

2019 ◽  
Vol 34 (8) ◽  
pp. 1608-1619 ◽  
Author(s):  
Heleen Masset ◽  
Masoud Zamani Esteki ◽  
Eftychia Dimitriadou ◽  
Jos Dreesen ◽  
Sophie Debrock ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Copy Number ◽  
Reference Data ◽  
Single Cells ◽  
Patent Application ◽  
Polymorphic Variant ◽  
Whole Genome ◽  
Reduced Representation ◽  
Allelic Frequencies ◽  
Genome Wide

Abstract STUDY QUESTION Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples? SUMMARY ANSWER Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples. WHAT IS KNOWN ALREADY Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology. STUDY DESIGN, SIZE, DURATION This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method. PARTICIPANTS/MATERIALS, SETTING, METHODS Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method. MAIN RESULTS AND THE ROLE OF CHANCE PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A). LIMITATIONS, REASONS FOR CAUTION Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases. WIDER IMPLICATIONS OF THE FINDINGS Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT. STUDY FUNDING/COMPETING INTEREST(S) Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 ‘Methods for haplotyping single cells’ and ZL913096-PCT/EP2014/068315 ‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 ‘High-throughput genotyping by sequencing’. Haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’) has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.

scholarly journals Erratum: Corrigendum: Genome-wide copy number analysis of single cells

2016 ◽  
Vol 11 (3) ◽  
pp. 616-616 ◽  
Author(s):  
Timour Baslan ◽  
Jude Kendall ◽  
Linda Rodgers ◽  
Hilary Cox ◽  
Mike Riggs ◽  
...  
Keyword(s):  
Copy Number ◽  
Single Cells ◽  
Copy Number Analysis ◽  
Genome Wide

scholarly journals Genome-wide copy number analysis of single cells

2012 ◽  
Vol 7 (6) ◽  
pp. 1024-1041 ◽  
Author(s):  
Timour Baslan ◽  
Jude Kendall ◽  
Linda Rodgers ◽  
Hilary Cox ◽  
Mike Riggs ◽  
...  
Keyword(s):  
Copy Number ◽  
Single Cells ◽  
Copy Number Analysis ◽  
Genome Wide

scholarly journals Low frequency somatic copy number alterations in normal human lymphocytes revealed by large scale single-cell whole genome profiling

2021 ◽  
Author(s):  
Lu Liu ◽  
He Chen ◽  
Cheng Sun ◽  
Jianyun Zhang ◽  
Juncheng Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Copy Number ◽  
Large Scale ◽  
Single Cells ◽  
Human Lymphocytes ◽  
Whole Genome ◽  
Copy Number Alterations ◽  
Monosomy X ◽  
Healthy Humans ◽  
Somatic Copy Number Alterations

Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations' stochastic natures. Using a Tn5-transposase assisted single-cell whole genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, while monosomy X occurred most frequently in over-30-year-old females. In the monosomy X single cells from individuals with phased genomes and identified X- inactivation ratios in bulk, the inactive X Chromosomes were lost more often than were the active ones.

Composite Single Cell Genetics and Clonal Phylogeny in Acute Lymphoblastic Leukaemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 122-122
Author(s):  
Nicola E Potter ◽  
Luca Ermini ◽  
Elli Papaemmanuil ◽  
Gowri Vijayaraghavan ◽  
Ian Titley ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Sequencing ◽  
Copy Number ◽  
Experimental Error ◽  
Genetic Architecture ◽  
Single Cells ◽  
Point Mutations ◽  
Whole Genome ◽  
Copy Number Alterations ◽  
Leukaemic Cells

Abstract Abstract 122 Cancer clone development is widely regarded as an evolutionary or Darwinian process of genetic diversification and natural (or therapeutic) selection within tissue ecosystems. Emerging studies are providing strong evidence that dynamic and complex branching sub-clonal genetic architectures are a common feature of cancer (Greaves M and Maley CC Nature 2012). This complexity may underpin the intransigence of advanced cancer to therapeutic control, particularly as the critical 'driver' cells – cancer or leukaemic stem cells, also appear to be genetically diverse within individual patients (Anderson K et al Nature 2011, Notta F et al Nature 2011). Sub-clonal architecture can only be fully determined through the study of large numbers of single cells uniformly sampled from the individual cancer of interest and assessed for composite genotype. Various technologies and approaches from fluorescent in situ hybridisation (FISH) to whole-genome sequencing of single cells have been applied to cancer and leukaemic cells but each approach has limitations. We have developed a novel multiplex microfluidic Q-PCR approach that allows unbiased single cell sampling, high throughput analysis of hundreds of individual cells and simultaneous detection of multiple genetic alterations in a single cell, including fusion genes, DNA copy number alterations (CNAs) and sequence-based mutations. As a proof of principle study we have applied this technique to REH, an acute lymphoblastic leukaemia (ALL) cell line that harbors the ETV6-RUNX1 fusion and a SNP in the EPO receptor gene, which we used as a surrogate mutation. We further determined a detailed sub-clonal genetic architecture for two ETV6-RUNX1 positive ALL patient samples with multiple point mutations and copy number alterations (determined by whole-genome sequencing) by interrogating approximately 400 flow cytometry sorted single cells with validation by FISH and standard sequencing. Briefly, single cells were lysed prior to multiplex specific (DNA) target amplification (STA) and Q-PCR using the 96.96 dynamic microfluidic array and the BioMarkï HD (Fluidigm, UK). Phylogenetic trees were constructed using maximum parsimony with PAUP analysis software. Interrogation of REH revealed that all single cells registered the ETV6-RUNX1 fusion and EPO receptor SNP, but 42% of cells gained either 1 or 2 additional copies of chromosome 21. Patient sample data revealed branching sub-clonal architectures in Case A in which all leukaemic cells harbored the fusion with additional point mutations but only sub-clones showed CNAs. In contrast, the sub-clonal architecture of Case B showed that whilst the ETV6-RUNX1 fusion was the earliest (or universal) genomic event, CNAs were relatively early events preceding the acquisition of point mutations (Figure 1). In both cases, the numerically predominant sub-clone harbored both point mutations and CNAs in addition to the presumptive initiating lesion, ETV6-RUNX1. These detailed and complex sub-clonal architectures would be masked by other genetic techniques. Single cell genetics coupled with deep genome sequencing is now technically feasible and provides an accurate portrait of the dynamic clonal complexity in leukaemia (and other cancers). Variegated genetics and clonal complexity in individual leukaemias has important implications for our understanding of molecular pathogenesis and for therapeutic targeting. Figure 1. This sub-clonal genetic architecture depicts the branching structure found for Case B, illustrating that in this case the ETV6-RUNX1 fusion was the earliest genomic event, followed by CNAs and the acquisition of point mutations. Those populations highlighted grey are within the experimental error rate but potentially true populations. Figure 1. This sub-clonal genetic architecture depicts the branching structure found for Case B, illustrating that in this case the ETV6-RUNX1 fusion was the earliest genomic event, followed by CNAs and the acquisition of point mutations. Those populations highlighted grey are within the experimental error rate but potentially true populations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PD2-2-7: Two wrongs make a right: the use of whole genome amplification for pair-wise genome-wide copy number analysis of limited patient material

2007 ◽  
Vol 2 (8) ◽  
pp. S442-S443
Author(s):  
Trevor J. Pugh ◽  
Allen D. Delaney ◽  
Stephane Flibotte ◽  
Noushin Farnoud ◽  
Irene Li ◽  
...  
Keyword(s):  
Copy Number ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Copy Number Analysis ◽  
Genome Amplification ◽  
Genome Wide

scholarly journals A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products

PLoS ONE ◽  
2018 ◽  
Vol 13 (3) ◽  
pp. e0193689 ◽  
Author(s):  
Alberto Ferrarini ◽  
Claudio Forcato ◽  
Genny Buson ◽  
Paola Tononi ◽  
Valentina del Monaco ◽  
...  
Keyword(s):  
Copy Number ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Whole Genome ◽  
Genome Amplification ◽  
Genome Wide ◽  
Low Pass

scholarly journals Accurate Genomic Variant Detection in Single Cells with Primary Template-Directed Amplification

2020 ◽  
Author(s):  
Veronica Gonzalez ◽  
Sivaraman Natarajan ◽  
Yuntao Xia ◽  
David Klein ◽  
Robert Carter ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Single Cell ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Variant Calling ◽  
Whole Genome ◽  
Genome Amplification ◽  
Genome Wide ◽  
Variant Detection ◽  
Genomic Variant

AbstractImprovements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here we present primary template-directed amplification (PTA), a new isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the new types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a new tool for mapping genome-wide interactions of mutagens with single living human cells at base pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate whole genome amplification, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

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