Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics

Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.

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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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SCCNV: a software tool for identifying copy number variation from single-cell whole-genome sequencing

10.1101/535807 ◽

2019 ◽

Cited By ~ 2

Author(s):

Xiao Dong ◽

Lei Zhang ◽

Xiaoxiao Hao ◽

Tao Wang ◽

Jan Vijg

Keyword(s):

Copy Number Variation ◽

Whole Genome Sequencing ◽

Single Cell ◽

Genome Sequencing ◽

Copy Number ◽

De Novo ◽

Single Cells ◽

Software Tool ◽

Whole Genome ◽

Number Variation

AbstractBackgroundIdentification of de novo mutations from cell populations requires single-cell whole-genome sequencing (SCWGS). Although many experimental protocols of SCWGS have been developed, few computational tools are available for downstream analysis of different types of somatic mutations, including copy number variation (CNV).ResultsWe developed SCCNV, a software tool for detecting CNVs from whole genome-amplified single cells. SCCNV is a read-depth based approach with adjustment for the whole-genome amplification bias.ConclusionsWe demonstrate its performance by analyzing data collected from most of the single-cell amplification methods, including DOP-PCR, MDA, MALBAC and LIANTI. SCCNV is freely available at https://github.com/biosinodx/SCCNV.

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Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

Malaria Journal ◽

10.1186/s12936-021-03630-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Noam B. Teyssier ◽

Anna Chen ◽

Elias M. Duarte ◽

Rene Sit ◽

Bryan Greenhouse ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Dropout Rate ◽

Dried Blood Spots ◽

High Ratio ◽

Low Density ◽

Whole Genome ◽

Cross Sectional ◽

Modest Improvement ◽

Primer Sets

Abstract Background Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. The effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification were evaluated on the quality and fidelity of WGS data recovered from DBS. Methods Low parasite density mock DBS samples were created, extracted either with Tween-Chelex or QIAamp, treated with or without McrBC, and amplified with one of three different amplification techniques (two sWGA primer sets and one rWGA). Extraction conditions were evaluated on performance of sequencing depth, percentiles of coverage, and expected SNP concordance. Results At 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5 × depth = 93% genome) than QIAamp extracted samples (5 × depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. Conclusions Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

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Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

10.21203/rs.3.rs-69257/v2 ◽

2020 ◽

Author(s):

Noam Teyssier ◽

Anna Chen ◽

Elias Duarte ◽

Rene Sit ◽

Bryan Greenhouse ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Dropout Rate ◽

Dried Blood Spots ◽

Low Density ◽

Whole Genome ◽

Cross Sectional ◽

Modest Improvement ◽

Blood Spots ◽

Gates Foundation

Abstract Background: Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. We evaluated the effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification on the quality and fidelity of WGS data recovered from DBS. Results: At 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5X depth = 93% genome) than QIAamp extracted samples (5X depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. Conclusions: Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys. Keywords: Malaria, P. falciparum, dried blood spots, Tween-Chelex, McrBC, selective whole genome amplification, whole genome sequencing This work was supported by the Bill & Melinda Gates Foundation, Grant Number OPP1132226 This work was supported by the Bill & Melinda Gates Foundation, Grant Number OPP1132226

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Publisher Correction: Amplification-free single-cell whole-genome sequencing gets a makeover

Nature Methods ◽

10.1038/s41592-020-0739-6 ◽

2020 ◽

Author(s):

Vesna Todorovic

Keyword(s):

Whole Genome Sequencing ◽

Single Cell ◽

Genome Sequencing ◽

Whole Genome

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Single-cell, whole-genome sequencing of patient tumor samples

Science-Business eXchange ◽

10.1038/scibx.2012.292 ◽

2012 ◽

Vol 5 (11) ◽

pp. 292-292

Keyword(s):

Whole Genome Sequencing ◽

Single Cell ◽

Genome Sequencing ◽

Whole Genome

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Integration of Whole-Genome Sequencing into Infection Control Practices: the Potential and the Hurdles

Journal of Clinical Microbiology ◽

10.1128/jcm.00349-15 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1054-1055 ◽

Cited By ~ 10

Author(s):

Elizabeth Robilotti ◽

Mini Kamboj

Keyword(s):

Infection Control ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Epidemiology ◽

Great Promise ◽

Whole Genome ◽

PaSD-qc: Quality control for single cell whole-genome sequencing data using power spectral density estimation

10.1101/166637 ◽

2017 ◽

Cited By ~ 2

Author(s):

Maxwell A. Sherman ◽

Alison R. Barton ◽

Michael Lodato ◽

Carl Vitzthum ◽

Michael E. Coulter ◽

...

Keyword(s):

Spectral Density ◽

Whole Genome Sequencing ◽

Single Cell ◽

Power Spectral Density ◽

Genome Sequencing ◽

Dna Amplification ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Power Spectral

AbstractSingle cell whole-genome sequencing (scWGS) is providing novel insights into the nature of genetic heterogeneity in normal and diseased cells. However, scWGS introduces DNA amplification-related biases that can confound downstream analysis. Here we present a statistical method, with an accompanying package PaSD-qc (Power Spectral Density-qc), that evaluates the quality of single cell libraries. It uses a modified power spectral density to assess amplification uniformity, amplicon size distribution, autocovariance, and inter-sample consistency as well as identifies aberrantly amplified chromosomes. We demonstrate the usefulness of this tool in evaluating scWGS protocols and in selecting high-quality libraries from low-coverage data for deep sequencing.

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Single cell whole genome sequencing reveals that NFKB1 mutation affects radiotherapy sensitivity in cervical cancer

Oncotarget ◽

10.18632/oncotarget.23587 ◽

2017 ◽

Vol 9 (7) ◽

pp. 7332-7340 ◽

Cited By ~ 2

Author(s):

Dong Yang ◽

Weiyuan Zhang ◽

JunQing Liang ◽

Kexin Ma ◽

Peng Chen ◽

...

Keyword(s):

Cervical Cancer ◽

Whole Genome Sequencing ◽

Single Cell ◽

Genome Sequencing ◽

Whole Genome

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Single-cell whole-genome sequencing reveals the functional landscape of somatic mutations in B lymphocytes across the human lifespan

10.1101/535906 ◽

2019 ◽

Cited By ~ 3

Author(s):

Lei Zhang ◽

Xiao Dong ◽

Moonsook Lee ◽

Alexander Y. Maslov ◽

Tao Wang ◽

...

Keyword(s):

B Cell ◽

Whole Genome Sequencing ◽

Single Cell ◽

B Lymphocytes ◽

Genome Sequencing ◽

Somatic Mutations ◽

Somatic Hypermutation ◽

Whole Genome ◽

Healthy Individuals ◽

Human Lifespan

Introductory paragraphThe accumulation of mutations in somatic cells have been implicated as a cause of ageing since the 1950s1,2. Yet, attempts to establish a causal relationship between somatic mutations and ageing have been constrained by the lack of methods to directly identify mutational events in primary human tissues. Here we provide detailed, genome-wide mutation frequencies and spectra of human B lymphocytes from healthy individuals across the entire human lifespan, from newborns to centenarians, using a recently developed, highly accurate single-cell whole-genome sequencing method3. We found that the number of somatic mutations increases from <500 per cell in newborns to >3,000 per cell in centenarians. We discovered mutational hotspot regions, some of which, as expected, located at immunoglobulin genes associated with somatic hypermutation. B cell-specific mutation signatures were observed associated with development, ageing or somatic hypermutation (SHM). The SHM signature strongly correlated with the signature found in human chronic lymphocytic leukemia and malignant B-cell lymphomas4, indicating that even in B cells of healthy individuals the potential cancer-causing events are already present. We also identified multiple mutations in sequence features relevant to cellular function, i.e., transcribed genes and gene regulatory regions. Such mutations increased significantly during ageing, but only at approximately half the rate of the genome average, indicating selection against mutations that impact B cell function. This first full characterization of the landscape of somatic mutations in human B lymphocytes indicates that spontaneous somatic mutations accumulating with age can be deleterious and may contribute to both the increased risk for leukemia and the functional decline of B lymphocytes in the elderly.

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