Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution.

DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97-0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.

Colorectal Cancer Stem Cell States Uncovered by Simultaneous Single-Cell Analysis of Transcriptome and Telomeres.

Cancer stem cells (CSCs) presumably contribute to tumor progression and drug resistance, yet their definitive features have remained elusive. Here, simultaneous measurement of telomere length and transcriptome in the same cells enables systematic assessment of CSCs in primary colorectal cancer (CRC). The in-depth transcriptome profiled by SMART-seq2 is independently validated by high-throughput scRNA-seq using 10 × Genomics. It is found that rare CSCs exist in dormant state and display plasticity toward cancer epithelial cells (EPCs) that essentially are presumptive tumor-initiating cells (TICs), while both retaining the prominent signaling pathways including WNT, TGF-ß, and HIPPO/YAP. Moreover, CSCs exhibit chromosome copy number variation (CNV) pattern resembling cancer EPCs but distinct from normal stem cells, suggesting the phylogenetic relationship between CSCs and cancer EPCs. Notably, CSCs maintain shorter telomeres and possess minimal telomerase activity consistent with their nonproliferative nature, unlike cancer EPCs. Additionally, the specific signature of CSCs particularly NOTUM, SMOC2, BAMBI, PHLDA1, and TNFRSF19 correlates with the prognosis of CRC. These findings characterize the heterogeneity of CSCs and their linkage to cancer EPCs/TICs, some of which are conventionally regarded as CSCs.

The Epigenetics of Gametes and Early Embryos and Potential Long-Range Consequences in Livestock Species-Filling in the Picture With Epigenomic Analyses.

The epigenome is dynamic and forged by epigenetic mechanisms, such as DNA methylation, histone modifications, chromatin remodeling, and non-coding RNA species. Increasing lines of evidence support the concept that certain acquired traits are derived from environmental exposure during early embryonic and fetal development, i.e., fetal programming, and can even be "memorized" in the germline as epigenetic information and transmitted to future generations. Advances in technology are now driving the global profiling and precise editing of germline and embryonic epigenomes, thereby improving our understanding of epigenetic regulation and inheritance. These achievements open new avenues for the development of technologies or potential management interventions to counteract adverse conditions or improve performance in livestock species. In this article, we review the epigenetic analyses (DNA methylation, histone modification, chromatin remodeling, and non-coding RNAs) of germ cells and embryos in mammalian livestock species (cattle, sheep, goats, and pigs) and the epigenetic determinants of gamete and embryo viability. We also discuss the effects of parental environmental exposures on the epigenetics of gametes and the early embryo, and evidence for transgenerational inheritance in livestock.

Magnesium is a critical element for competent development of bovine embryos.

The study was designed to determine the impact of magnesium (Mg2+) on bovine embryo development. We found that two commercially available sources of bovine serum albumin (BSA) and fetal bovine serum (FBS) contained different amounts of Mg2+ residue: 4 ppm in ICPbio BSA, 114 ppm in Sigma BSA, and 44 ppm in FBS. When CR1 was used as basal medium, PVA and ICPbio BSA produced the lowest blastocyst yield (2.2-2.3%), whereas Sigma BSA increased blastocyst yield to 18.9% (P < 0.05). Supplementation of 1.4 mM MgCl2 into the medium increased the blastocyst rate in the ICPbio BSA group (29.4%) but not in the PVA group (5.4%; P < 0.05) to a level comparable to that of the FBS group (33.7%; P > 0.05). We next found that increasing concentrations of MgCl2 in the culture medium (ICPbio BSA) elevated blastocyst rate from 2.6% (0 mM), 38.4% (0.35 mM) to 50.2% (1.4 mM; P < 0.05), further maintained at 44.9% (2.1 mM) and 43.4% (2.8 mM) (P > 0.05). However, blastocyst rate was reduced to 31.4% (4.2 mM) and 29.4% (5.6 mM) when MgCl2 supplement was increased (P < 0.05). Comparable blastocyst development was achieved in both ICPbio BSA (30.0-33.1%) and Sigma BSA (37.4-38.7%) groups when 1.4 mM Mg2+ was supplemented regardless of its source (MgCl2 vs. MgSO4; P > 0.05). In embryo transfer experiments, higher rates of pregnancy (54.3 vs. 41.5%) and calving (44.3 vs. 32.5%) were achieved in the CR1-Mg2+-supplemented BSA group compared with the FBS group with co-culture, respectively (P < 0.05). These results demonstrate that Mg2+ is a key ion that promotes competent blastocyst and term development. Therefore, a simple and efficient defined medium (CR1-Mg2+-BSA) can successfully replace complex serum and somatic cell co-culture.

Methylome Dynamics of Bovine Gametes and in vivo Early Embryos.

DNA methylation undergoes drastic fluctuation during early mammalian embryogenesis. The dynamics of global DNA methylation in bovine embryos, however, have mostly been studied by immunostaining. We adopted the whole genome bisulfite sequencing (WGBS) method to characterize stage-specific genome-wide DNA methylation in bovine sperm, immature oocytes, oocytes matured in vivo and in vitro, as well as in vivo developed single embryos at the 2-, 4-, 8-, and 16-cell stages. We found that the major wave of genome-wide DNA demethylation was complete by the 8-cell stage when de novo methylation became prominent. Sperm and oocytes were differentially methylated in numerous regions (DMRs), which were primarily intergenic, suggesting that these non-coding regions may play important roles in gamete specification. DMRs were also identified between in vivo and in vitro matured oocytes, suggesting environmental effects on epigenetic modifications. In addition, virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, by using RNA-seq data generated from embryos at the same developmental stages, we revealed a weak inverse correlation between gene expression and promoter methylation. This comprehensive analysis provides insight into the critical features of the bovine embryo methylome, and serves as an important reference for embryos produced in vitro, such as by in vitro fertilization and cloning. Lastly, these data can also provide a model for the epigenetic dynamics in human early embryos.

DNA methylomes of bovine gametes and in vivo produced preimplantation embryos.

DNA methylation is an important epigenetic modification that undergoes dynamic changes in mammalian embryogenesis, during which both parental genomes are reprogrammed. Despite the many immunostaining studies that have assessed global methylation, the gene-specific DNA methylation patterns in bovine preimplantation embryos are unknown. Using reduced representation bisulfite sequencing, we determined genome-scale DNA methylation of bovine sperm and individual in vivo developed oocytes and preimplantation embryos. We show that (1) the major wave of genome-wide demethylation was completed by the 8-cell stage; (2) promoter methylation was significantly and inversely correlated with gene expression at the 8-cell and blastocyst stages; (3) sperm and oocytes have numerous differentially methylated regions (DMRs)-DMRs specific for sperm were strongly enriched in long terminal repeats and rapidly lost methylation in embryos; while the oocyte-specific DMRs were more frequently localized in exons and CpG islands (CGIs) and demethylated gradually across cleavage stages; (4) DMRs were also found between in vivo and in vitro matured oocytes; and (5) differential methylation between bovine gametes was confirmed in some but not all known imprinted genes. Our data provide insights into the complex epigenetic reprogramming of bovine early embryos, which serve as an important model for human preimplantation development.

Characterization of cancer genomic heterogeneity by next-generation sequencing advances precision medicine in cancer treatment.

Cancer is a heterogeneous disease with unique genomic and phenotypic features that differ between individual patients and even among individual tumor regions. In recent years, large-scale genomic studies and new next-generation sequencing technologies have uncovered more scientific details about tumor heterogeneity, with significant implications for the choice of specific molecular biomarkers and clinical decision making. Genomic heterogeneity significantly contributes to the generation of a diverse cell population during tumor development and progression, representing a determining factor for variation in tumor treatment response. It has been considered a prominent contributor to therapeutic failure, and increases the likelihood of resistance to future therapies in most common cancers. The understanding of molecular heterogeneity in cancer is a fundamental component of precision oncology, enabling the identification of genomic alteration of key genes and pathways that can be targeted therapeutically. Here, we review the emerging knowledge of tumor genomics and heterogeneity, as well as potential implications for precision medicine in cancer treatment and new therapeutic discoveries. An analysis and interpretation of the TCGA database was included.

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.

Next-generation molecular diagnosis: single-cell sequencing from bench to bedside.

Single-cell sequencing (SCS) is a fast-growing, exciting field in genomic medicine. It enables the high-resolution study of cellular heterogeneity, and reveals the molecular basis of complicated systems, which facilitates the identification of new biomarkers for diagnosis and for targeting therapies. It also directly promotes the next generation of genomic medicine because of its ultra-high resolution and sensitivity that allows for the non-invasive and early detection of abnormalities, such as aneuploidy, chromosomal translocation, and single-gene disorders. This review provides an overview of the current progress and prospects for the diagnostic applications of SCS, specifically in pre-implantation genetic diagnosis/screening, non-invasive prenatal diagnosis, and analysis of circulating tumor cells. These analyses will accelerate the early and precise control of germline- or somatic-mutation-based diseases, particularly single-gene disorders, chromosome abnormalities, and cancers.

Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects.

Advances in genomic technology have enabled the faithful detection and measurement of mutations and the gene expression profile of cancer cells at the single-cell level. Recently, several single-cell sequencing methods have been developed that permit the comprehensive and precise analysis of the cancer-cell genome, transcriptome, and epigenome. The use of these methods to analyze cancer cells has led to a series of unanticipated discoveries, such as the high heterogeneity and stochastic changes in cancer-cell populations, the new driver mutations and the complicated clonal evolution mechanisms, and the novel identification of biomarkers of variant tumors. These methods and the knowledge gained from their utilization could potentially improve the early detection and monitoring of rare cancer cells, such as circulating tumor cells and disseminated tumor cells, and promote the development of personalized and highly precise cancer therapy. Here, we discuss the current methods for single cancer-cell sequencing, with a strong focus on those practically used or potentially valuable in cancer research, including single-cell isolation, whole genome and transcriptome amplification, epigenome profiling, multi-dimensional sequencing, and next-generation sequencing and analysis. We also examine the current applications, challenges, and prospects of single cancer-cell sequencing.

Effects of High Hydrostatic Pressure on Expression Profiles of In Vitro Produced Vitrified Bovine Blastocysts.

High hydrostatic pressure (HHP) has been used to pre-condition embryos before essential, yet potentially detrimental procedures such as cryopreservation. However, the mechanisms for HHP are poorly understood. We treated bovine blastocysts with three different HHP (40, 60 and 80 MPa) in combination with three recovery periods (0, 1 h, 2 h post HHP). Re-expansion rates were significantly higher at 40 and 60 but lower at 80 MPa after vitrification-warming in the treated groups than controls. Microarray analysis revealed 399 differentially expressed transcripts, representing 254 unique genes, among different groups. Gene ontology analysis indicated that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes in cell death and apoptosis, and up-regulation of genes in RNA processing, cellular growth and proliferation. In contrast, 80 MPa up-regulated genes in apoptosis, and down-regulated protein folding and cell cycle-related genes. Moreover, gene expression was also influenced by the length of the recovery time after HHP. The significantly over-represented categories were apoptosis and cell death in the 1 h group, and protein folding, response to unfolded protein and cell cycle in the 2 h group compared to 0 h. Taken together, HHP promotes competence of vitrified bovine blastocysts through modest transcriptional changes.

mRNA Levels of Imprinted Genes in Bovine In Vivo Oocytes, Embryos and Cross Species Comparisons with Humans, Mice and Pigs.

Twenty-six imprinted genes were quantified in bovine in vivo produced oocytes and embryos using RNA-seq. Eighteen were detectable and their transcriptional patterns were: largely decreased (MEST and PLAGL1); first decreased and then increased (CDKN1C and IGF2R); peaked at a specific stage (PHLDA2, SGCE, PEG10, PEG3, GNAS, MEG3, DGAT1, ASCL2, NNAT, and NAP1L5); or constantly low (DIRAS3, IGF2, H19 and RTL1). These patterns reflect mRNAs that are primarily degraded, important at a specific stage, or only required at low quantities. The mRNAs for several genes were surprisingly abundant. For instance, transcripts for the maternally imprinted MEST and PLAGL1, were high in oocytes and could only be expressed from the maternal allele suggesting that their genomic imprints were not yet established/recognized. Although the mRNAs detected here were likely biallelically transcribed before the establishment of imprinted expression, the levels of mRNA during these critical stages of development have important functional consequences. Lastly, we compared these genes to their counterparts in mice, humans and pigs. Apart from previously known differences in the imprinting status, the mRNA levels were different among these four species. The data presented here provide a solid reference for expression profiles of imprinted genes in embryos produced using assisted reproductive biotechnologies.

Two methods for full-length RNA sequencing for low quantities of cells and single cells.

The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3' biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling.

Indistinguishable transcriptional profiles between in vitro- and in vivo-produced bovine fetuses.

During the past several decades, in vitro fertilization (IVF) has been increasingly used both in animal production and human infertility treatment. Animals derived from in vitro manipulation are occasionally associated with abnormal offspring syndrome (AOS) and other developmental abnormalities. By studying gene expression of in vitro-produced (IVP) embryos/animals, we gain an indicator of how well this procedure mimics the in vivo environment. Most previous studies of this nature have focused on only a few genes at a time or have been limited to studying the pre-implantation stage; a global view of how gene transcription may be influenced by in vitro procedures during fetal development has yet to be ascertained. To this end, we collected liver and placental tissue samples from IVP and in vivo control bovine fetuses at days 90 and 180 of gestation. We used a bovine 13K oligonucleotide microarray to investigate the transcriptional profiles in both tissues from IVP fetuses, and compared them with those of their age-matched in vivo counterparts. Surprisingly, in both liver and placental tissues, the transcriptional profiles between IVP and control fetuses, at either 90 or 180 days of gestation, were indistinguishable. A total of 879 genes were found to be significantly regulated during liver development from 90 to 180 days of gestation, but there were no gene expression changes in the placental tissue during this developmental period. Quantitative real-time RT-PCR on 11 selected genes confirmed these results. Our results have certain implications for IVF technologies, both in agriculture and in human medicine.

[Technological advances in single-cell genomic analyses].

The technological progress of the genomics has transformed life science research. The main objectives of genomics are sequencing of new genomes and genome-wide identification of the function and the interaction of genes and their products. The recently developed second generation or next generation sequencing platforms and DNA microarray technology are immensely important and powerful tools for functional genomic analyses. However, their application is limited by the requirement of sufficient amounts of high quality nucleic acid samples. Therefore, when only a single cell or a very small number of cells are available or are preferred, the whole genomic sequencing or functional genomic objectives cannot be achieved conventionally and require a robust amplification method. This review highlights DNA amplification technologies and summarizes the strategies currently utilized for whole genome sequencing of a single cell, with specific focus on studies investigating microorganisms; An outline for targeted re-sequencing enabling the analysis of larger genomes is also provided. Furthermore, the review presents the emerging functional genomic applications using next-generation sequencing or microarray analysis to examine genome-wide transcriptional profile, chromatin modification and other types of protein-DNA binding profile, and CpG methylation mapping in a single cell or a very low quantity of cells. The nature of these technologies and their prospects are also addressed.

Transferase activity function and system development process are critical in cattle embryo development.

Microarray gene expression experiments often consider specific developmental stages, tissue sources, or reproductive technologies. This focus hinders the understanding of the cattle embryo transcriptome. To address this, four microarray experiments encompassing three developmental stages (7, 25, 280 days), two tissue sources (embryonic or extra-embryonic), and two reproductive technologies (artificial insemination or AI and somatic cell nuclear transfer or NT) were combined using two sets of meta-analyses. The first set of meta-analyses uncovered 434 genes differentially expressed between AI and NT (regardless of stage or source) that were not detected by the individual-experiment analyses. The molecular function of transferase activity was enriched among these genes that included ECE2, SLC22A1, and a gene similar to CAMK2D. Gene POLG2 was over-expressed in AI versus NT 7-day embryos and was under-expressed in AI versus NT 25-day embryos. Gene HAND2 was over-expressed in AI versus NT extra-embryonic samples at 280 days yet under-expressed in AI versus NT embryonic samples at 7 days. The second set of meta-analyses uncovered enrichment of system, organ, and anatomical structure development among the genes differentially expressed between 7- and 25-day embryos from either reproductive technology. Genes PRDX1and SLC16A1 were over-expressed in 7- versus 25-day AI embryos and under-expressed in 7- versus 25-day NT embryos. Changes in stage were associated with high number of differentially expressed genes, followed by technology and source. Genes with transferase activity may hold a clue to the differences in efficiency between reproductive technologies.

Comprehensive annotation of the transcriptome of the human fungal pathogen Candida albicans using RNA-seq.

Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections that are often lifethreatening. We have used massively parallel high-throughput sequencing of cDNA (RNA-seq) to generate a high-resolution map of the C. albicans transcriptome under several different environmental conditions. We have quantitatively determined all of the regions that are transcribed under these different conditions, and have identified 602 novel transcriptionally active regions (TARs) and numerous novel introns that are not represented in the current genome annotation. Interestingly, the expression of many of these TARs is regulated in a condition-specific manner. This comprehensive transcriptome analysis significantly enhances the current genome annotation of C. albicans, a necessary framework for a complete understanding of the molecular mechanisms of pathogenesis for this important eukaryotic pathogen.

Altered gene expression profiles in the brain, kidney, and lung of deceased neonatal cloned pigs.

Limited studies have been published analyzing the gene expression patterns of cloned pigs. We compared the expression profiles of brain, kidney, and lung tissues, representing each of the three germ layers, of deceased neonatal cloned pigs with those of age-matched controls using a 13K oligonucleotide microarray. We found 42 (0.7% of total genes analyzed), 178 (2.9%), and 121 (1.9%) genes differentially expressed in the brain, kidney, and lung of clones, respectively, when compared with the corresponding organs from controls (fold change >1.5, p < 0.05, false discovery rate (FDR) = 0.05). These expression aberrations could potentially cause the following pathological anomalies in clones: diabetic nephropathy in the kidney and dysregulated surfactant homeostasis in the lung. Interestingly, upregulated expression of genes belonging to the MAPK pathway was observed in all three organs. To investigate whether the differences in levels of gene expression were caused by differential DNA methylation, the global DNA methylation level was measured by high-performance liquid chromatography. In controls, global concentration of methylated cytosine was 5.35%, whereas clones had significantly hypomethylated genomic DNA (4.57%). Bisulfite-pyrosequencing analyses of the promoter regions of differentially expressed candidate genes, c-MYC, Period 1 (PER1), Cathepsin L (CTSL), and Follistatin (FS), however, did not show any differences in the degree of DNA methylation between controls and clones. Our findings demonstrate that deceased neonatal cloned pigs have considerable gene expression abnormalities, which may have contributed to the death of the animals.

Embryonic gene expression profiling using microarray analysis.

Microarray technology enables the interrogation of thousands of genes at one time and therefore a systems level of analysis. Recent advances in the amplification of RNA, genome sequencing and annotation, and the lower cost of developing microarrays or purchasing them commercially, have facilitated the analysis of single preimplantation embryos. The present review discusses the components of embryonic expression profiling and examines current research that has used microarrays to study the effects of in vitro production and nuclear transfer.