Construction of thousands of single cell genome sequencing libraries using combinatorial indexing

Vitak, Sarah A; Torkenczy, Kristof A.; Rosenkrantz, Jimi L.; Fields, Andrew J.; Christiansen, Lena; Wong, Melissa H.; Carbone, Lucia; Steemers, Frank J.; Adey, Andrew

doi:10.1101/065482

Cited by 2 publications

(4 citation statements)

References 32 publications

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“…Finally, HIPSD-seq can also be combined with an additional combinatorial indexing step [46] to achieve ultra-high throughput scDNA-seq (sci-HIPSD-seq, > 10,000 cells) (Methods). Data from all three protocols can be processed using the standard Cell Ranger pipelines.…”

Section: Resultsmentioning

confidence: 99%

“…The first scDNA-seq protocols have been extended and improved in different ways, to increase coverage and reduce bias (the latter through amplification-free workflows for example) [26,[40][41][42] and to allow for parallel profiling of DNA and RNA from the same cells [36,37,[42][43][44]. Most recently, with the advent of combinatorial indexing, the feasibility of scaling scDNA-seq to tens of thousands of cells in a single experiment has been demonstrated [46]. However, what is lacking to date are schemes that render high-throughput scDNA-seq accessible and reproducible across laboratories.…”

Section: Introductionmentioning

confidence: 99%

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HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling

Lazareva,

Mallm,

Simovic-Lorenz

et al. 2023

Preprint

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Single-cell DNA-sequencing (scDNA-seq) enables decoding somatic cancer variation. Existing methods are hampered by low throughput or cannot be combined with transcriptome sequencing in the same cell. We propose HIPSD&R-seq (HIgh-throughPutSingle-cellDna andRna-seq), a scalable yet simple assay to profile low-coverage DNA and RNA in thousands of cells in parallel. Our approach builds on an accessible modification of the 10X Genomics platform for scATAC and multiome profiling. In applications to human cell models and primary tissue, we demonstrate the feasibility to detect rare clones and we combine the assay with combinatorial indexing to profile over 16,000 cells.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling

Lazareva,

Mallm,

Simovic-Lorenz

et al. 2023

Preprint

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“…In the cell nucleus, more than 96% genomic DNA is inaccessible to enzymes and proteins besides histone. 15,16 To detect the inaccessible NAMs in single cells, one requires the physical compartmentalization of different single cells and their genomic DNA after cell lysis. The single-cell genomic DNA must be exposed without cross-contamination and subjected to the following multistep reactions mainly including chemical labeling and DNA amplification.…”

Section: Single-cell Hydrogel Encoding Amplification To Detect Global...mentioning

confidence: 99%

“…On the other hand, only about 4% of genomic DNA in native chromatins is dynamically accessible to proteins or enzymes. 15,16 The NAMs existing in inaccessible DNA may also play significant roles. These multilayered features of NAMs may vary in different single cells (Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

Lighting Up Nucleic Acid Modifications in Single Cells with DNA-Encoded Amplification

et al. 2022

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Metrics & More Article Recommendations CONSPECTUS: Nucleic acids are naturally decorated with various chemical modifications at nucleobases. Most nucleic acid modifications (NAMs) do not alter Watson−Crick base pairing but can regulate gene expression known as "epigenetics". Their abundances present a very wide range, approximately 10 −2 to 10 −6 of total bases. Different NAMs may coexist in spatial proximity (e.g., <20 nm) in the crowded intracellular environment. Considering the highly dynamic chromatin accessibility (physical access to DNA), the NAMs in inaccessible DNA probably plays different roles. These multilayered features of NAMs vary from cell to cell. Our understanding of the function and mechanism of NAMs in biological processes and disease states has largely been driven by the expanding array of sequencing-based methodologies. However, an underexplored aspect is the measurement of the subcellular distribution, spatial proximity, and inaccessibility of NAMs in single cells. In recent years, we have developed new approaches that light up single-cell NAMs with single-site sensitivity. These methods are mainly based on the integration of chemical or chemoenzymatic tools, DNA amplification and nanotechnology, and/or microfluidics. An overview of these methods together with conventional methods such as immunofluorescence (IF) and fluorescence in situ hybridization (FISH) is provided in this Account. Our laboratory has proposed DNA-encoded amplification (DEA) as the main strategy for developing a set of single-cell NAM imaging methods. In brief, DEA transforms the different features of NAMs into unique DNA primers for rolling circle amplification (RCA) followed by FISH imaging. The first method is base-encoded amplifying FISH (BEA-FISH), in which we convert individual NAMs into RCA primers via chemoselective labeling and click bioconjugation. It enables the in situ visualization of low-abundanceNAMs (e.g., 5hmU), which is impracticable by conventional methods. We subsequently developed pairwise proximity-differentiated amplifying FISH (PPDA-FISH), which integrates BEA-FISH with DNA nanotechnology. PPDA-FISH utilizes proximity ligation and toehold strand displacement to label the adjacent site of two different NAMs (one-to-one proximity) and their respective residual sites with three unique RCA probes. It achieves simultaneous counting of the above-mentioned three types of modified sites in the same cells. The third method is cellular macromolecule-tethered DNA walking indexing (Cell-TALKING) to probe more than two NAMs within the same nanoenvironments. Cell-TALKING uses dynamic DNA proximity cleavage to continuously activate different preblocked RCA primers (for each NAM) near one walking probe (for one target molecule). We have explored three NAMs around one histone (one-to-many proximity) in different cancer cell lines and clinical specimens. Then, we describe a single-cell hydrogel encoding amplification (scHEA) method by integrating droplet microfluidics with BEA-FISH. This method generates hydrogel be...

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Construction of thousands of single cell genome sequencing libraries using combinatorial indexing

Cited by 2 publications

References 32 publications

HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling

HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling

Lighting Up Nucleic Acid Modifications in Single Cells with DNA-Encoded Amplification

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