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

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.

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Detection and characterization of copy number variants based on whole-genome sequencing by DNBSEQ platforms

10.1101/786962 ◽

2019 ◽

Author(s):

Junhua Rao ◽

Lihua Peng ◽

Fang Chen ◽

Hui Jiang ◽

Chunyu Geng ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Copy Number ◽

Copy Number Variants ◽

Copy Number Variant ◽

Whole Genome ◽

Genome Wide ◽

Wide Range ◽

Distribution Sensitivity ◽

Cnv Detection

AbstractBackgroundNext-generation sequence (NGS) has rapidly developed in past years which makes whole-genome sequencing (WGS) becoming a more cost- and time-efficient choice in wide range of biological researches. We usually focus on some variant detection via WGS data, such as detection of single nucleotide polymorphism (SNP), insertion and deletion (Indel) and copy number variant (CNV), which playing an important role in many human diseases. However, the feasibility of CNV detection based on WGS by DNBSEQ™ platforms was unclear. We systematically analysed the genome-wide CNV detection power of DNBSEQ™ platforms and Illumina platforms on NA12878 with five commonly used tools, respectively.ResultsDNBSEQ™ platforms showed stable ability to detect slighter more CNVs on genome-wide (average 1.24-fold than Illumina platforms). Then, CNVs based on DNBSEQ™ platforms and Illumina platforms were evaluated with two public benchmarks of NA12878, respectively. DNBSEQ™ and Illumina platforms showed similar sensitivities and precisions on both two benchmarks. Further, the difference between tools for CNV detection was analyzed, and indicated the selection of tool for CNV detection could affected the CNV performance, such as count, distribution, sensitivity and precision.ConclusionThe major contribution of this paper is providing a comprehensive guide for CNV detection based on WGS by DNBSEQ™ platforms for the first time.

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

Neuro-Oncology ◽

10.1093/neuonc/noaa222.563 ◽

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.

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

Nucleic Acids Research ◽

10.1093/nar/gkz717 ◽

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.

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Genome‐wide detection of copy number variants in European autochthonous and commercial pig breeds by whole‐genome sequencing of DNA pools identified breed‐characterising copy number states

Animal Genetics ◽

10.1111/age.12954 ◽

2020 ◽

Vol 51 (4) ◽

pp. 541-556

Author(s):

S. Bovo ◽

A. Ribani ◽

M. Muñoz ◽

E. Alves ◽

J. P. Araujo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Copy Number ◽

Copy Number Variants ◽

Whole Genome ◽

Pig Breeds ◽

Genome Wide ◽

Dna Pools

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Aberrant replication licensing drives Copy Number Gains across species

10.1101/2020.10.10.334516 ◽

2020 ◽

Author(s):

Patroula Nathanailidou ◽

Michalis Petropoulos ◽

Styliani Maxouri ◽

Eirini Kasselimi ◽

Ioanna Eleni Symeonidou ◽

...

Keyword(s):

Drug Resistance ◽

Genetic Analysis ◽

Genome Sequencing ◽

Genetic Heterogeneity ◽

Copy Number ◽

Mammalian Cells ◽

Whole Genome ◽

Genome Wide ◽

Repair Pathways ◽

Replication Intermediates

AbstractCopy Number Gains (CNGs) lead to genetic heterogeneity, driving evolution and carcinogenesis. The mechanisms promoting CNG formation however remain poorly characterized. We show that abnormal expression of the replication licensing factor Cdc18 in fission yeast, which leads to genome-wide re-replication, drives the formation of CNGs at different genomic loci, promoting the acquisition of new selectable traits. Whole genome sequencing reveals Mb long, primarily extrachromosomal amplicons. Genetic analysis shows that homology-mediated repair is required to resolve re-replication intermediates into heritable CNGs. Consistently, we show that in mammalian cells overexpression of CDC6 and/or CDT1 leads to CNGs and promotes drug resistance. In human cells, multiple repair pathways are activated upon rereplication and act antagonistically, with RAD52 promoting and 53BP1 inhibiting CNG formation. In tumours, CDT1 and/or CDC6 overexpression correlates with copy number gains genome-wide. We propose re-replication as an evolutionary-conserved driver of CNGs, highlighting a link between aberrant licensing, CNGs and cancer.

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Composite Single Cell Genetics and Clonal Phylogeny in Acute Lymphoblastic Leukaemia

Blood ◽

10.1182/blood.v120.21.122.122 ◽

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.

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

PLoS ONE ◽

10.1371/journal.pone.0193689 ◽

2018 ◽

Vol 13 (3) ◽

pp. e0193689 ◽

Cited By ~ 8

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

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

10.1101/564914 ◽

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).

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Global characterization of copy number variants in epilepsy patients from whole genome sequencing

10.1101/199224 ◽

2017 ◽

Author(s):

Jean Monlong ◽

Simon L. Girard ◽

Caroline Meloche ◽

Maxime Cadieux-Dion ◽

Danielle M. Andrade ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Copy Number ◽

Copy Number Variants ◽

Genetic Variations ◽

Genome Wide Association Studies ◽

Whole Genome ◽

Loss Of Function ◽

Genome Wide

AbstractEpilepsy will affect nearly 3% of people at some point during their lifetime. Previous copy number variants (CNVs) studies of epilepsy have used array-based technology and were restricted to the detection of large or exonic events. In contrast, whole-genome sequencing (WGS) has the potential to more comprehensively profile CNVs but existing analytic methods suffer from limited accuracy. We show that this is in part due to the non-uniformity of read coverage, even after intra-sample normalization. To improve on this, we developed PopSV, an algorithm that uses multiple samples to control for technical variation and enables the robust detection of CNVs. Using WGS and PopSV, we performed a comprehensive characterization of CNVs in 198 individuals affected with epilepsy and 301 controls. For both large and small variants, we found an enrichment of rare exonic events in epilepsy patients, especially in genes with predicted loss-of-function intolerance. Notably, this genome-wide survey also revealed an enrichment of rare non-coding CNVs near previously known epilepsy genes. This enrichment was strongest for non-coding CNVs located within 100 Kbp of an epilepsy gene and in regions associated with changes in the gene expression, such as expression QTLs or DNase I hypersensitive sites. Finally, we report on 21 potentially damaging events that could be associated with known or new candidate epilepsy genes. Our results suggest that comprehensive sequence-based profiling of CNVs could help explain a larger fraction of epilepsy cases.Author summaryEpilepsy is a common neurological disorder affecting around 3% of the population. In some cases, epilepsy is caused by brain trauma or other brain anomalies but there are often no clear causes. Genetic factors have been associated with epilepsy in the past such as rare genetic variations found by linkage studies as well as common genetic variations found by genome-wide association studies and large copy-number variants. We sequenced the genome of ∼200 epilepsy patients and ∼300 healthy controls and compared the distribution of deletion (loss of a copy) and duplication (additional copy) of genomic regions. Thanks to the sequencing technology and a new method that takes advantage of the large sample size, we could compare the distribution of small copy- number variants between epilepsy patients and controls. Overall, we found that small variants are also associated with epilepsy. Indeed, the genome of epilepsy patients had more exonic copy- number variants, especially when rare or affecting genes with predicted loss-of-function intolerance. Focusing on regions around genes that have been previously associated with epilepsy, we also found more non-coding variants in epilepsy patients, especially deletions or variants in regulatory regions. Finally, we provide a list of 21 regions in which we found likely pathogenic variants.

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