scSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

Raine, Amanda; Lundmark, Anders; Annett, Alva; Wiman, Ann-Christin; Cavalli, Marco; Wadelius, Claes; Bergin, Claudia; Nordlund, Jessica

doi:10.1038/s41598-022-09798-2

Cited by 8 publications

(4 citation statements)

References 33 publications

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“…This technique uses a randomer-annealed splint oligo to ligate the desired adapter, along with the addition of single-stranded DNA binding (SSB) protein to enhance efficiency. A similar approach was also recently described for direct adapter ligation for single-cell DNA methylation profiling, scSPLAT 7 , though without the use of SSB and leveraging different adapter designs. After ligation, indexed PCR is performed prior to pooling and sequencing.…”

Section: Resultsmentioning

confidence: 99%

High-throughput robust single-cell DNA methylation profiling with sciMETv2

et al. 2022

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DNA methylation is a key epigenetic property that drives gene regulatory programs in development and disease. Current single-cell methods that produce high quality methylomes are expensive and low throughput without the aid of extensive automation. We previously described a proof-of-principle technique that enabled high cell throughput; however, it produced only low-coverage profiles and was a difficult protocol that required custom sequencing primers and recipes and frequently produced libraries with excessive adapter contamination. Here, we describe a greatly improved version that generates high-coverage profiles (~15-fold increase) using a robust protocol that does not require custom sequencing capabilities, includes multiple stopping points, and exhibits minimal adapter contamination. We demonstrate two versions of sciMETv2 on primary human cortex, a high coverage and rapid version, identifying distinct cell types using CH methylation patterns. These datasets are able to be directly integrated with one another as well as with existing snmC-seq2 datasets with little discernible bias. Finally, we demonstrate the ability to determine cell types using CG methylation alone, which is the dominant context for DNA methylation in most cell types other than neurons and the most applicable analysis outside of brain tissue.

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Section: Resultsmentioning

confidence: 99%

High-throughput robust single-cell DNA methylation profiling with sciMETv2

et al. 2022

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“…Paired ICM and TE blastocyst samples ( n = 24) were processed and analyzed using the ultra-low DNA input whole genome bisulfite sequencing (WGBS) prep workflow (Zymo Research, Irvine, CA). Samples were prepared for WGBS using an adapted library preparation protocol [ 28 ]. Approximately 0.5 pg of methylation sequencing in situ spike-in control oligos were added to help validate and assess the overall workflow and resulting data output.…”

Section: Methodsmentioning

confidence: 99%

Paternal aging impacts expression and epigenetic markers as early as the first embryonic tissue lineage differentiation

Denomme,

McCallie,

Haywood

et al. 2024

Hum Genomics

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Background Advanced paternal age (APA) is associated with adverse outcomes to offspring health, including increased risk for neurodevelopmental disorders. The aim of this study was to investigate the methylome and transcriptome of the first two early embryonic tissue lineages, the inner cell mass (ICM) and the trophectoderm (TE), from human blastocysts in association with paternal age and disease risk. High quality human blastocysts were donated with patient consent from donor oocyte IVF cycles from either APA (≥ 50 years) or young fathers. Blastocysts were mechanically separated into ICM and TE lineage samples for both methylome and transcriptome analyses. Results Significant differential methylation and transcription was observed concurrently in ICM and TE lineages of APA-derived blastocysts compared to those from young fathers. The methylome revealed significant enrichment for neuronal signaling pathways, as well as an association with neurodevelopmental disorders and imprinted genes, largely overlapping within both the ICM and TE lineages. Significant enrichment of neurodevelopmental signaling pathways was also observed for differentially expressed genes, but only in the ICM. In stark contrast, no significant signaling pathways or gene ontology terms were identified in the trophectoderm. Despite normal semen parameters in aged fathers, these significant molecular alterations can adversely contribute to downstream impacts on offspring health, in particular neurodevelopmental disorders like autism spectrum disorder and schizophrenia. Conclusions An increased risk for neurodevelopmental disorders is well described in children conceived by aged fathers. Using blastocysts derived from donor oocyte IVF cycles to strategically control for maternal age, our data reveals evidence of methylation dysregulation in both tissue lineages, as well as transcription dysregulation in neurodevelopmental signaling pathways associated with APA fathers. This data also reveals that embryos derived from APA fathers do not appear to be compromised for initial implantation potential with no significant pathway signaling disruption in trophectoderm transcription. Collectively, our work provides insights into the complex molecular mechanisms that occur upon paternal aging during the first lineage differentiation in the preimplantation embryo. Early expression and epigenetic markers of APA-derived preimplantation embryos highlight the susceptibility of the future fetus to adverse health outcomes.

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“…Single-cell splinted ligation adapter tagging (scSPLAT) improves mappability using a splinted adapter as the second adapter. No artificial low-complexity sequence is added, and the process is free from the risk of artificial sequences due to the carryover of free nucleotides 32 .…”

Section: Objectives Of Single-cell Mono-omicsmentioning

confidence: 99%

Advances in single-cell omics and multiomics for high-resolution molecular profiling

Lim,

Park,

Kim

et al. 2024

Exp Mol Med

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Single-cell omics technologies have revolutionized molecular profiling by providing high-resolution insights into cellular heterogeneity and complexity. Traditional bulk omics approaches average signals from heterogeneous cell populations, thereby obscuring important cellular nuances. Single-cell omics studies enable the analysis of individual cells and reveal diverse cell types, dynamic cellular states, and rare cell populations. These techniques offer unprecedented resolution and sensitivity, enabling researchers to unravel the molecular landscape of individual cells. Furthermore, the integration of multimodal omics data within a single cell provides a comprehensive and holistic view of cellular processes. By combining multiple omics dimensions, multimodal omics approaches can facilitate the elucidation of complex cellular interactions, regulatory networks, and molecular mechanisms. This integrative approach enhances our understanding of cellular systems, from development to disease. This review provides an overview of the recent advances in single-cell and multimodal omics for high-resolution molecular profiling. We discuss the principles and methodologies for representatives of each omics method, highlighting the strengths and limitations of the different techniques. In addition, we present case studies demonstrating the applications of single-cell and multimodal omics in various fields, including developmental biology, neurobiology, cancer research, immunology, and precision medicine.

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scSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

Cited by 8 publications

References 33 publications

High-throughput robust single-cell DNA methylation profiling with sciMETv2

High-throughput robust single-cell DNA methylation profiling with sciMETv2

Paternal aging impacts expression and epigenetic markers as early as the first embryonic tissue lineage differentiation

Advances in single-cell omics and multiomics for high-resolution molecular profiling

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