Single-cell genome sequencing: current state of the science.

Chapter 17 - Robotics in Single-Cell Omics

Single microbial cell genome sequencing is becoming a powerful tool for the discovery of the hidden genetic information valuable for many medical applications. One of the critical steps in single-cell genome sequencing is the physical isolation of individual cells from a highly diverse heterogeneous population. Amplifying the genome of a single microbial cell is another challenge due to the minute amount of DNA. Efforts have been directed in developing an optofluidic platform integrating advanced microscopy, optical tweezers and microfluidic technology for single cell isolation and genome amplification. Here, we investigate and evaluate the validity of this platform for single microbial cell genome amplification. The successful validation of this approach allows us to perform various single cell studies using this platform.

SUMMARYSingle-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge of current methods is their inability to identify and provide accurate quantitative information for low-abundance proteins. Herein, we describe an ion-mobility-enhanced mass spectrometry acquisition and peptide identification method, transferring identification based on FAIMS filtering (TIFF), to improve the sensitivity and accuracy of label-free scProteomics. TIFF extends the ion accumulation times for peptide ions by filtering out singly charged ions. The peptide identities are assigned by a three-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells, with >1,100 proteins consistently identified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during lipopolysaccharide stimulation and identified time-dependent proteome changes. A record of this paper’s transparent peer review process is included in the supplemental information.

Copy number alterations (CNAs) play an important role in molding the genomes of breast cancers and have been shown to be clinically useful for prognostic and therapeutic purposes. However, our knowledge of intra-tumoral genetic heterogeneity of this important class of somatic alterations is limited. Here, using single-cell sequencing, we comprehensively map out the facets of copy number alteration heterogeneity in a cohort of breast cancer tumors. Ou/var/www/html/elife/12-05-2020/backup/r analyses reveal: genetic heterogeneity of non-tumor cells (i.e. stroma) within the tumor mass; the extent to which copy number heterogeneity impacts breast cancer genomes and the importance of both the genomic location and dosage of sub-clonal events; the pervasive nature of genetic heterogeneity of chromosomal amplifications; and the association of copy number heterogeneity with clinical and biological parameters such as polyploidy and estrogen receptor negative status. Our data highlight the power of single-cell genomics in dissecting, in its many forms, intra-tumoral genetic heterogeneity of CNAs, the magnitude with which CNA heterogeneity affects the genomes of breast cancers, and the potential importance of CNA heterogeneity in phenomena such as therapeutic resistance and disease relapse.

Copy number alterations (CNAs) play an important role in molding the genomes of breast cancers and have been shown to be clinically useful for prognostic and therapeutic purposes. However, our knowledge of intra-tumoral genetic heterogeneity of this important class of somatic alterations is limited. Here, using single-cell sequencing, we comprehensively map out the facets of copy number alteration heterogeneity in a cohort of breast cancer tumors. Ou/var/www/html/elife/12-05-2020/backup/r analyses reveal: genetic heterogeneity of non-tumor cells (i.e. stroma) within the tumor mass; the extent to which copy number heterogeneity impacts breast cancer genomes and the importance of both the genomic location and dosage of sub-clonal events; the pervasive nature of genetic heterogeneity of chromosomal amplifications; and the association of copy number heterogeneity with clinical and biological parameters such as polyploidy and estrogen receptor negative status. Our data highlight the power of single-cell genomics in dissecting, in its many forms, intra-tumoral genetic heterogeneity of CNAs, the magnitude with which CNA heterogeneity affects the genomes of breast cancers, and the potential importance of CNA heterogeneity in phenomena such as therapeutic resistance and disease relapse.

The advent and development of single-cell whole-genome sequencing (scWGS) technology has shed lights on the genomic heterogeneities within biosamples at the single-cell resolution. The technology is particularly well-established in the recent decade and witnesses a variety of clinical applications, such as circulating tumor cell (CTC) detection and preimplantation genetic diagnosis/screening (PGD/PGS). In this review, we summarize the latest practical breakthroughs of scWGS in the field of biomedicine, with the hope of providing a guideline to apply single-cell genomic sequencing in clinical researches.

Single biological molecule studies enable to probe and visualize exciting details of the events in physiological in vivo processes. The basic underlying question of this dissertation is to understand biological processes at a single molecule level. In contrast to ensemble techniques, advances in single molecule manipulation (e.g. optical and magnetic tweezers, atomic force microscopy) and / or fluorescence techniques allow to investigate the properties of individual molecules in real time with a possibility to change external conditions (buffers) in situ and modulate inter- and intra-molecular interactions. This thesis reports the application of a single molecule technique, dual beam optical tweezers, for the study of single biomolecules. A range of single molecule systems was investigated such as i)VirE2 protein DNA machinery, ii) DNA-surfactant, EtBr (ethidium bromide), SYBR® Green-DNA interactions and iii) dsDNA denaturation studies. In addition the development of the present experimental setup is described to enable combined force measurement as well as single molecule fluorescence studies. The presented biomolecular results provide new and complementary information on the different biological systems demonstrating the diversity of experiments that can be performed on single DNA molecules using optical tweezers. Chapter one gives a brief introduction to optical tweezers, describes how optical tweezers work, the physics behind it, details of the experimental setup and the method of force calibration required in micromanipulation. Optical tweezers have opened exciting avenues of research, especially in biology. Biologists will be able to investigate the nature of molecular machines one by one, and infer from their behavior those properties common to the population. In chapter 2, we show how optical tweezers were employed to study the change in the mechanical properties of single DNA molecules upon binding of small agents. The first part of this chapter reports on the changes in mechanics of single dsDNA in the presence of cationic and anionic surfactants (used as non-viral vectors in gene therapy). The second part describes the interaction of DNA binding ligands (SYBR® Green, EtBr) with individual DNA strands. Agrobacterium tumefaciens (AT), a Gram-negative bacterium, evolved a complex and unique mechanism to transfer a long single stranded DNA (ssDNA) molecule from its cytoplasm to the eukaryotic host plant cell nucleus. Central to this mechanism, chapter 3 discusses the results of the measurements on VirE2 protein interacting with single stranded DNA (ssDNA). VirE2 protein is a multifunctional protein from AT that coat the transferred-ssDNA (T-DNA), interacts with host factors assisting nuclear import of the complex, forms channels in lipid bilayers and displays a highly cooperative binding to ssDNA. The biological findings are presented in a new generic model which can be used to explain how generation of forces helps bacterial DNA to enter the plant cell based on our single molecule data. Single molecule dsDNA denaturation, relevant in many molecular biological experiments, induced by NaOH and mechanical pulling are studied in chapter 4. Here optical tweezers experiments give access to the ‘melting’ of hydrogen bonds by mechanical forces or alkali denaturation (NaOH) of dsDNA in real time. The mechanical stability and the transition of dsDNA to ssDNA is investigated at different ionic strength as well as in buffers. Fluorescent images of single λ DNA labeled with SYBR® Green were observed up to forces ≥ 65 pN and indicate a B-DNA to S −DNA transition. Chapter 5 describes the implementation of single-molecule fluorescence detection (SMF) in optical tweezers. The design and instrumental capabilities of optical tweezers combined with SMF are discussed in detail. The development of this instrument provides a worldwide unique experimental setup and opens up new possibilities in the studies of complex biological systems. Finally chapter 6 summarizes the results of this thesis and discusses future experimental applications. The appendices provide further details for DNA sample preparation, molecular biology and chemical surface activation recipes, an instruction manual for the setup and the list of currently published papers.

Optofluidic single-cell genome amplification was used to obtain genome sequences from sub-micron cells collected from the euphotic and mesopelagic zones of the northwestern Sargasso Sea. Plankton cells were visually selected and manually sorted with an optical trap, yielding 20 partial genome sequences representing seven bacterial phyla. Two organisms, E01-9C-26 (Gammaproteobacteria), represented by four single cell genomes, and Opi.OSU.00C, an uncharacterized Verrucomicrobia, were the first of their types retrieved by single cell genome sequencing and were studied in detail. Metagenomic data showed that E01-9C-26 is found throughout the dark ocean, while Opi.OSU.00C was observed to bloom transiently in the nutrient-depleted euphotic zone of the late spring and early summer. The E01-9C-26 genomes had an estimated size of 4.76–5.05 Mbps, and contained “O” and “W”-type monooxygenase genes related to methane and ammonium monooxygenases that were previously reported from ocean metagenomes. Metabolic reconstruction indicated E01-9C-26 are likely versatile methylotrophs capable of scavenging C1 compounds, methylated compounds, reduced sulfur compounds, and a wide range of amines, including D-amino acids. The genome sequences identified E01-9C-26 as a source of “O” and “W”-type monooxygenase genes related to methane and ammonium monooxygenases that were previously reported from ocean metagenomes, but are of unknown function. In contrast, Opi.OSU.00C genomes encode genes for catabolizing carbohydrate compounds normally associated with eukaryotic phytoplankton. This exploration of optofluidics showed that it was effective for retrieving diverse single-cell bacterioplankton genomes and has potential advantages in microbiology applications that require working with small sample volumes or targeting cells by their morphology.

<p dir="ltr">Cancer is a highly heterogeneous disease, both at the genomic and phenotypic level. Single-cell methods have allowed exploration of phenotypic cell states and genomic evolution, but the association between genotype and phenotype remains largely elusive. Activating or deleterious single nucleotide variants (SNVs) can have very clear effects on a phenotype, but copy number variations (CNVs), which are ubiquitous in cancers, are not well understood functionally. Recent advances within ultra-low coverage, single-cell whole genome sequencing (WGS) have enabled high throughput analysis of CNV. However, downstream analysis is still largely dependent on tools developed for high coverage bulk data.</p><p dir="ltr">In <b>paper I</b> we developed a computational method, ASCENT, which enables accurate breakpoint detection, absolute copy number calling, and haplotyping of clonal segments. We showed that by using ASCENT we could find minor subclones not detected in bulk WGS data, and produce accurate high-resolution copy number profiles from ultra-low coverage single-cell WGS.</p><p dir="ltr">By running ASCENT on joint mRNA/WGS data from 57 patients representing six cancer types, we were able to make generalizable inferences about how subclonal genetics affect cell phenotypes (<b>paper III</b>). We found that highly amplified CNVs constrain the phenotype to a much greater degree than lowly amplified whole chromosome aneuploidies. We found that gene dosage is largely tissue-dependent and that oncogenes, such as MYC, are often not sensitive to dosage. We identified a previously underappreciated group of tumors that lack a clear clonal structure, where each cell division leads to two distinct genotypes.</p><p dir="ltr">In <b>paper IV</b> we analyzed the relationship between genotype and phenotype during induction treatment in pediatric acute lymphoblastic leukemia (ALL). We found that while some genotypes have specific phenotypes, during induction treatment cell states shifted toward a more mature B-cell-like state, independent of genomic background. We contrast this to the mechanism which leads to relapse, which always includes additional genomic aberrations, selection on the genetic level, and inter-patient heterogeneity in cell states.</p><p dir="ltr">Single-cell sequencing methods are often used to determine which cell states are responsible for phenotypes found in bulk sequencing. In <b>paper II</b> we investigated the complicated relationship between shear stress and vascular disease in the aorta. We used single-cell sequencing to deconvolve which cell type caused an immune-pro phenotype in AmotL2-depleted mice and found a subset of endothelial cells to be responsible.</p><p dir="ltr">In conclusion, we have charted the transcriptional effect of different genetic aberrations across human cancer. We conclude that specific genomic aberrations can affect the functional phenotype of a cell. We found that dosage effects are largely tissue-dependent and that the amplification of oncogenes is often compensated for on the mRNA-level. We additionally found that induction treatment confers a common cell state in ALL, while relapsed samples had evolved divergent phenotypes during low intensity treatment.</p><h3 dir="ltr">List of scientific papers</h3><p dir="ltr">I. <b>Solrun Kolbeinsdottir</b>*, Vasilios Zachariadis*, Christian Sommerauer, Olli Lohi, Merja Heinäniemi, Martin Enge. Absolute copy number aware CNV calling of sub megabase segments in ultra-low coverage single-cell DNA sequencing data. Nucleic Acids Research, Volume 53, Issue 17, 23 September 2025. <a href="https://doi.org/10.1093/nar/gkaf919" rel="noreferrer" target="_blank">https://doi.org/10.1093/nar/gkaf919</a></p><p dir="ltr">II. Yuanyuan Zhang, Yumeng Zhang, Evelyn Hutterer, Sara Hultin, Otto Bergman, <b>Solrun Kolbeinsdottir</b>, Hong Jin, Maria J Forteza, Daniel F J Ketelhuth, Joy Roy, Ulf Hedin, Martin Enge, Ljubica Matic, Per Eriksson, Lars Holmgren. The VE-cadherin/AmotL2 mechanosensory pathway suppresses aortic inflammation and the formation of abdominal aortic aneurysms. Nature Cardiovascular Research, 2023, 7, 629-644. <a href="https://doi.org/10.1038/s44161-023-00298-8" rel="noreferrer" target="_blank">https://doi.org/10.1038/s44161-023-00298-8</a></p><p dir="ltr">III. <b>Solrun Kolbeinsdottir</b>, Vasilios Zachariadis, Muyi Yang, Luuk Broeils, Christian Sommerauer, Huaitao Cheng, Xinsong Chen, Yingbo Lin, Sampsa Hautaniemi, Johanna Hynninen, Suzanne Egyhazi Brage, Dhifaf Sarhan, Anna Vähärautio, Nikolas Herold, Johan Hartman, Hildur Helgadóttir, Felix Haglund de Flon, Martin Enge. Subclonal copy number alterations and their transcriptional impacts across human cancers using joint single-cell genome and transcriptome sequencing. [Manuscript]</p><p dir="ltr">IV. Vasilios Zachariadis, <b>Solrun Kolbeinsdottir</b>, Jessica Hacheney, Huaitao Cheng, Laura Oksa, Aonghus Naughton, Arghavan Alizadeh, Sanni Moisio, Olli Lohi, Merja Heinäniemi, Martin Enge. Persister states and relapse in childhood leukemia. [Manuscript]</p><p dir="ltr">*These authors contributed equally</p>

<p dir="ltr">Cancer is a highly heterogeneous disease, both at the genomic and phenotypic level. Single-cell methods have allowed exploration of phenotypic cell states and genomic evolution, but the association between genotype and phenotype remains largely elusive. Activating or deleterious single nucleotide variants (SNVs) can have very clear effects on a phenotype, but copy number variations (CNVs), which are ubiquitous in cancers, are not well understood functionally. Recent advances within ultra-low coverage, single-cell whole genome sequencing (WGS) have enabled high throughput analysis of CNV. However, downstream analysis is still largely dependent on tools developed for high coverage bulk data.</p><p dir="ltr">In <b>paper I</b> we developed a computational method, ASCENT, which enables accurate breakpoint detection, absolute copy number calling, and haplotyping of clonal segments. We showed that by using ASCENT we could find minor subclones not detected in bulk WGS data, and produce accurate high-resolution copy number profiles from ultra-low coverage single-cell WGS.</p><p dir="ltr">By running ASCENT on joint mRNA/WGS data from 57 patients representing six cancer types, we were able to make generalizable inferences about how subclonal genetics affect cell phenotypes (<b>paper III</b>). We found that highly amplified CNVs constrain the phenotype to a much greater degree than lowly amplified whole chromosome aneuploidies. We found that gene dosage is largely tissue-dependent and that oncogenes, such as MYC, are often not sensitive to dosage. We identified a previously underappreciated group of tumors that lack a clear clonal structure, where each cell division leads to two distinct genotypes.</p><p dir="ltr">In <b>paper IV</b> we analyzed the relationship between genotype and phenotype during induction treatment in pediatric acute lymphoblastic leukemia (ALL). We found that while some genotypes have specific phenotypes, during induction treatment cell states shifted toward a more mature B-cell-like state, independent of genomic background. We contrast this to the mechanism which leads to relapse, which always includes additional genomic aberrations, selection on the genetic level, and inter-patient heterogeneity in cell states.</p><p dir="ltr">Single-cell sequencing methods are often used to determine which cell states are responsible for phenotypes found in bulk sequencing. In <b>paper II</b> we investigated the complicated relationship between shear stress and vascular disease in the aorta. We used single-cell sequencing to deconvolve which cell type caused an immune-pro phenotype in AmotL2-depleted mice and found a subset of endothelial cells to be responsible.</p><p dir="ltr">In conclusion, we have charted the transcriptional effect of different genetic aberrations across human cancer. We conclude that specific genomic aberrations can affect the functional phenotype of a cell. We found that dosage effects are largely tissue-dependent and that the amplification of oncogenes is often compensated for on the mRNA-level. We additionally found that induction treatment confers a common cell state in ALL, while relapsed samples had evolved divergent phenotypes during low intensity treatment.</p><h3 dir="ltr">List of scientific papers</h3><p dir="ltr">I. <b>Solrun Kolbeinsdottir</b>*, Vasilios Zachariadis*, Christian Sommerauer, Olli Lohi, Merja Heinäniemi, Martin Enge. Absolute copy number aware CNV calling of sub megabase segments in ultra-low coverage single-cell DNA sequencing data. Nucleic Acids Research, Volume 53, Issue 17, 23 September 2025. <a href="https://doi.org/10.1093/nar/gkaf919" rel="noreferrer" target="_blank">https://doi.org/10.1093/nar/gkaf919</a></p><p dir="ltr">II. Yuanyuan Zhang, Yumeng Zhang, Evelyn Hutterer, Sara Hultin, Otto Bergman, <b>Solrun Kolbeinsdottir</b>, Hong Jin, Maria J Forteza, Daniel F J Ketelhuth, Joy Roy, Ulf Hedin, Martin Enge, Ljubica Matic, Per Eriksson, Lars Holmgren. The VE-cadherin/AmotL2 mechanosensory pathway suppresses aortic inflammation and the formation of abdominal aortic aneurysms. Nature Cardiovascular Research, 2023, 7, 629-644. <a href="https://doi.org/10.1038/s44161-023-00298-8" rel="noreferrer" target="_blank">https://doi.org/10.1038/s44161-023-00298-8</a></p><p dir="ltr">III. <b>Solrun Kolbeinsdottir</b>, Vasilios Zachariadis, Muyi Yang, Luuk Broeils, Christian Sommerauer, Huaitao Cheng, Xinsong Chen, Yingbo Lin, Sampsa Hautaniemi, Johanna Hynninen, Suzanne Egyhazi Brage, Dhifaf Sarhan, Anna Vähärautio, Nikolas Herold, Johan Hartman, Hildur Helgadóttir, Felix Haglund de Flon, Martin Enge. Subclonal copy number alterations and their transcriptional impacts across human cancers using joint single-cell genome and transcriptome sequencing. [Manuscript]</p><p dir="ltr">IV. Vasilios Zachariadis, <b>Solrun Kolbeinsdottir</b>, Jessica Hacheney, Huaitao Cheng, Laura Oksa, Aonghus Naughton, Arghavan Alizadeh, Sanni Moisio, Olli Lohi, Merja Heinäniemi, Martin Enge. Persister states and relapse in childhood leukemia. [Manuscript]</p><p dir="ltr">*These authors contributed equally</p>

991013049328803412 HKUST Electronic Theses Molecular technology for single-cell genome and transcriptome paired sequencing by Loi Shek Chun Danson thesis 2022 1 online resource (x, 89 pages) : illustrations (some color) Single-cell sequencing has allowed the non-averaged measurements of genotypic and…Read more ›

Single-cell sequencing is a novel approach to genome sequencing of clinical samples. However, there are only few studies using single-cell sequencing of genomes for bacterial infections. A 71-year-old woman presented to the emergency department with epigastric pain, 38.5°C fever, and a history of hypertension and hyperuricemia. From blood test results, acute cholecystitis was suspected. The surgery went well and bilirubin calcium stones were found in the gallbladder. Single-cell sequencing was used to investigate a bile sample from a patient with acute cholecystitis. The sample, cultured on a MacConkey agar plate, produced four colonies, all identified as Escherichia coli by bacteriological and biochemical properties. Whole genome sequences of the four strains were determined using the single-cell amplified genome (SAG) sequencing technique. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of all four were 99.98-100% and 100%, respectively, indicating that they were the same bacterial species. Compared with type strains, these four strains were closest to Shigella sonnei (ANI 98.65-98.66%; dDDH 88.5%) than E. coli (ANI 96.79-96.80%; dDDH 74.2%), despite lacking stx1, stx2 and ipaH, which Shigella species harbor. 16S metagenome analysis identified E. coli as the predominant bacterial genome in the sample, comprising 93.15%. SAG raw data had a relatively high level of quality, with 98.4-98.7% of the read numbers used after quality trimming. However, the genome sequencing coverage was only 9.45-42.88% when compared to a complete genome of an isolate with a mapping quality set above 99%, resulting in gaps compared to conventional whole genome sequence data of these isolates. The procedures of the SAG sequencing technique should be revised to improve the sequencing coverage and reduce gaps in the sequence data. Nonetheless, single-cell genome sequencing can provide novel information for bacterial infections.

Genome sequencing has uncovered a new mutational phenomenon in cancer and congenital disorders called chromothripsis. Chromothripsis is characterized by extensive genomic rearrangements and an oscillating pattern of DNA copy number levels, all curiously restricted to one or a few chromosomes. The mechanism for chromothripsis is unknown, but we previously proposed that it could occur through the physical isolation of chromosomes in aberrant nuclear structures called micronuclei. Here, using a combination of live-cell imaging and single-cell genome sequencing, we demonstrate that micronucleus formation can indeed generate a spectrum of genomic rearrangements, some of which recapitulate all known features of chromothripsis. These events are restricted to the missegregated chromosome and occur within one cell division. We demonstrate that the mechanism for chromothripsis can involve the fragmentation and subsequent reassembly of a single chromatid from a micronucleus. Collectively, these experiments establish a new mutational process of which chromothripsis is one extreme outcome.

To understand the role of the skin commensal bacterial community in skin health and the spread of pathogens, it is crucial to identify genetic differences in the bacterial strains corresponding to human individuals. A culture-independent genomics approach is an effective tool for obtaining massive high-quality bacterial genomes. Here we present a single-cell genome sequencing to obtain comprehensive whole-genome sequences of uncultured skin bacteria from skin swabs. We recovered 281 high-quality (HQ) and 244 medium-quality single-amplified genomes (SAGs) of multiple skin bacterial species from eight individuals, including cohabiting group. Single-cell sequencing outperformed in the genome recovery from the same skin swabs, showing 10-fold non-redundant strain genomes compared to the shotgun metagenomic sequencing and binning approach. We then focused on the abundant skin bacteria and identified intra-species diversity, especially in 47 Moraxella osloensis derived HQ SAGs, characterizing the strain-level heterogeneity at mobile genetic element profiles, including plasmids and prophages. Even between the cohabiting individual hosts, they have unique skin bacterial strains in the same species, which shows microdiversity in each host. Genetic and functional differences between skin bacterial strains are predictive of in vivo competition to adapt bacterial genome to utilize the sparse nutrients available on the skin or produce molecules that inhibit the colonization of other microbes or alter their behavior. Thus, single-cell sequencing provides a large number of genomes of higher resolution and quality than conventional metagenomic analysis and helps explore the skin commensal bacteria at the strain level, linking taxonomic and functional information.

We describe a method for in situ sizing individual huge DNA molecules by laser trapping. Single DNA molecules are reversibly transformed, without mechanical fragmentation of fragile huge-sized DNA, from their random coil state into their globular state induced by condensing agents poly(ethylene glycol) and Mg(2+). With the use of a globular DNA molecule folded by condensation, the critical velocity of the circularly accelerated single globular DNA molecule by laser trapping was found to be proportional to the size of the DNA. Yeast, Saccharomyces cerevisiae, chromosome III (285 kbp) was successfully sized (281 +/- 40 kbp) from a calibration curve scaled using lambda, T4, and yeast chromosome VI (48.5, 166, and 385 kbp, respectively). The use of critical velocity as a sizing parameter makes it possible to size single DNA molecules without prior conformational information, i.e., the radius of a single globular huge DNA molecule as a nanoparticle. A sized single globular DNA molecule could be trapped again for subsequent manipulation, such as transportation of it anywhere. We also investigated a possibility of reusing the globular DNA molecules condensed by PEG and Mg(2+) for PCR and found that PCR efficiency was not deteriorated in the presence of the condensation agents.