Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI)

Chen, Chongyi; Xing, Dong; Tan, Longzhi; Li, Heng; Zhou, Guangyu; Huang, Lei; Xie, Xiaohui

doi:10.1126/science.aak9787

Cited by 329 publications

(306 citation statements)

References 64 publications

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“…6 Investigating biological activities at the single-cell level can reveal novel regulatory mechanisms for diverse cellular processes, which has significant implication in the field of biomedicine. 3 In fact, in recent years researchers have performed single-cell studies to investigate various biological issues, including the epigenetic regulation of single cells, 7 single-cell extraction for molecular analyses, 8 single-cell whole-genome analyses, 9 single-cell dynamics, 10 and stem cell behaviors, 11 providing novel ways to address biological issues in the life sciences. Down to the single-cell level, it is known that the fulfillment of cell functions is based on a complex set of molecular interactions which control signaling pathways to influence ultimate cell fates, including proliferation, differentiation, and apoptosis.…”

Section: Introductionmentioning

confidence: 99%

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

Dang

et al. 2017

Nanoscale

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Due to the lack of adequate tools for observation, native molecular behaviors at the nanoscale have been poorly understood. The advent of atomic force microscopy (AFM) provides an exciting instrument for investigating physiological processes on individual living cells with molecular resolution, which attracts the attention of worldwide researchers. In the past few decades, AFM has been widely utilized to investigate molecular activities on diverse biological interfaces, and the performances and functions of AFM have also been continuously improved, greatly improving our understanding of the behaviors of single molecules in action and demonstrating the important role of AFM in addressing biological issues with unprecedented spatiotemporal resolution. In this article, we review the related techniques and recent progress about applying AFM to characterize biomolecular systems in situ from single molecules to living cells. The challenges and future directions are also discussed.

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Section: Introductionmentioning

confidence: 99%

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

Dang

et al. 2017

Nanoscale

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“…This method requires sensitive whole genome amplification techniques (5); however, DNA bases are susceptible to damage (which can lead to mutation), and enzymes used in amplification introduce additional errors. In fact, these false-positive single nucleotide mutations (also referred to as SNVs) can be as high as 10 4 in single-cell WGS (5), vastly outnumbering naturally-occurring SNVs (10 2 –10 3 /cell). Even in cancer or population genome sequencing projects, mutagenic DNA damage can be a major source of sequencing error for rare variants (6).…”

mentioning

confidence: 99%

“…The accuracy of single-cell WGS continues to improve, including new amplification methods with a lower error rate and more uniform coverage of the genome (5). Unfortunately, single-cell WGS is limited to a small number of cells due to throughput and cost bottlenecks.…”

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confidence: 99%

Tracing single-cell histories

Lee

2018

Science

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“…http://dx.doi.org/10.1101/158352 doi: bioRxiv preprint first posted online Jun. 30, 2017; Dileep and Gilbert, page 3Single-cell DNA copy number can distinguish replicated DNA from un-replicated DNA 15,16 . Specifically, regions that have completed replication will have twice the copy number compared to regions that have not replicated.…”

mentioning

confidence: 99%

“…Single-cell DNA copy number can distinguish replicated DNA from un-replicated DNA 15,16 . Specifically, regions that have completed replication will have twice the copy number compared to regions that have not replicated.…”

mentioning

confidence: 99%

Single-cell replication profiling reveals stochastic regulation of the mammalian replication-timing program

Dileep¹,

Dm²

2017

Preprint

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In mammalian cells, distinct replication domains (RDs), corresponding to structural units of chromosomes called topologically-associating domains (TADs), replicate at different times during S-phase [1][2][3][4] . Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments 5,6 . Although replication origins are selected stochastically, such that each cell is using a different cohort of origins to replicate their genomes [7][8][9][10][11][12] , replication-timing is regulated independently and upstream of origin selection 13 and evidence suggests that replication timing is conserved in consecutive cell cycles 14 . Hence, quantifying the extent of cell-to-cell variation in replication timing is central to studies of chromosome structure and function. Here we devise a strategy to measure variation in single-cell replication timing using DNA copy number. We find that borders between replicated and un-replicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus.Nonetheless, measurable variation was evident. Surprisingly, we detected a similar degree of variation in replication timing from cell-to-cell, between homologues within cells, and between all domains genome-wide regardless of their replication timing.These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/158352 doi: bioRxiv preprint first posted online Jun. 30, 2017; Dileep and Gilbert, page 3Single-cell DNA copy number can distinguish replicated DNA from un-replicated DNA 15,16 . Specifically, regions that have completed replication will have twice the copy number compared to regions that have not replicated. Hence, we reasoned that measurements of DNA copy number in cells isolated at different times during S-phase could reveal replication-timing programs in single-cells. Moreover, to separately evaluate the extent of extrinsic (cell-to-cell) vs. intrinsic (homologue-to-homologue) variability in replication timing, we examined both haploid H129-2 mouse embryonic stem cells (mESCs) and diploid hybrid musculus 129 × Castaneus mESCs that harbor a high single nucleotide polymorphism (SNP) density between homologues, permitting allele specific analysis. To generate single-cell copy number variation (CNV) profiles, we used flow cytometry of DNA-stained cells to sort single S-phase cells into 96 well plates followed by whole genome amplification (WGA). Amplified DNA from each cell was uniquely barcoded and sequenced (Fig. 1a) 17,18 . To control for amplification and mappability biases, we also sorted G1 and G2 cells, which contain a uniform DNA content. Regions of low mappability and over amplification were removed based on the G1 and G2 controls. Read counts were normalized by dividing the co...

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Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI)

Cited by 329 publications

References 64 publications

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

Tracing single-cell histories

Single-cell replication profiling reveals stochastic regulation of the mammalian replication-timing program

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