Single-cell RNA-seq analysis of mouse preimplantation embryos by third-generation sequencing

The development of next generation sequencing (NGS) platform-based single-cell RNA sequencing (scRNA-seq) techniques has tremendously changed biological researches, while there are still many questions that cannot be addressed by them due to their short read lengths. We developed a novel scRNA-seq technology based on third-generation sequencing (TGS) platform (single-cell amplification and sequencing of full-length RNAs by Nanopore platform, SCAN-seq). SCAN-seq exhibited high sensitivity and accuracy comparable to NGS platform-based scRNA-seq methods. Moreover, we captured thousands of unannotated transcripts of diverse types, with high verification rate by reverse transcription PCR (RT-PCR)–coupled Sanger sequencing in mouse embryonic stem cells (mESCs). Then, we used SCAN-seq to analyze the mouse preimplantation embryos. We could clearly distinguish cells at different developmental stages, and a total of 27,250 unannotated transcripts from 9,338 genes were identified, with many of which showed developmental stage-specific expression patterns. Finally, we showed that SCAN-seq exhibited high accuracy on determining allele-specific gene expression patterns within an individual cell. SCAN-seq makes a major breakthrough for single-cell transcriptome analysis field.

[1]  Roger Volden,et al.  Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2 , 2020, bioRxiv.

[2]  Pascal Barbry,et al.  High throughput error corrected Nanopore single cell transcriptome sequencing , 2019, Nature Communications.

[3]  F. Tang,et al.  Human Germline Cell Development: from the Perspective of Single-Cell Sequencing. , 2019, Molecular cell.

[4]  M. Robinson,et al.  A comprehensive examination of Nanopore native RNA sequencing for characterization of complex transcriptomes , 2019, Nature Communications.

[5]  Yasuko Mori,et al.  Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen , 2019, Nature Communications.

[6]  Lu Wen,et al.  Single-Cell Transcriptome Analysis Maps the Developmental Track of the Human Heart. , 2019, Cell reports.

[7]  A. Khamlichi,et al.  Parallels between Mammalian Mechanisms of Monoallelic Gene Expression. , 2018, Trends in genetics : TIG.

[8]  Ahmed Mahfouz,et al.  Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells , 2018, Nature Biotechnology.

[9]  F. Luciani,et al.  High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes , 2018, Nature Communications.

[10]  Richard E. Green,et al.  Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA , 2018, Proceedings of the National Academy of Sciences.

[11]  G. Lettre,et al.  PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells , 2018, BMC Medical Genetics.

[12]  S. Orkin,et al.  Mapping the Mouse Cell Atlas by Microwell-Seq , 2018, Cell.

[13]  Jie Qiao,et al.  A single-cell RNA-seq survey of the developmental landscape of the human prefrontal cortex , 2018, Nature.

[14]  Jia Gu,et al.  fastp: an ultra-fast all-in-one FASTQ preprocessor , 2018, bioRxiv.

[15]  Onur Mutlu,et al.  Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions , 2017, Briefings Bioinform..

[16]  Mauricio O. Carneiro,et al.  Scaling accurate genetic variant discovery to tens of thousands of samples , 2017, bioRxiv.

[17]  Heng Li,et al.  Minimap2: pairwise alignment for nucleotide sequences , 2017, Bioinform..

[18]  Jing He,et al.  Allelic reprogramming of 3D chromatin architecture during early mammalian development , 2017, Nature.

[19]  Rong Li,et al.  Single-Cell RNA-Seq Analysis Maps Development of Human Germline Cells and Gonadal Niche Interactions. , 2017, Cell stem cell.

[20]  Hugh E. Olsen,et al.  Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells , 2017, Nature Communications.

[21]  Lennart Martens,et al.  SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification , 2017, bioRxiv.

[22]  S. Antonarakis,et al.  Detection of Imprinted Genes by Single-Cell Allele-Specific Gene Expression. , 2017, American journal of human genetics.

[23]  Botond Sipos,et al.  Highly parallel direct RNA sequencing on an array of nanopores , 2016, Nature Methods.

[24]  Jeffrey T Leek,et al.  Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown , 2016, Nature Protocols.

[25]  F. Tang,et al.  Single-cell sequencing in stem cell biology , 2016, Genome Biology.

[26]  I. Hellmann,et al.  Comparative Analysis of Single-Cell RNA Sequencing Methods , 2016, bioRxiv.

[27]  F. Tang,et al.  Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos , 2015, Genome Biology.

[28]  B. Herrmann,et al.  Long noncoding RNAs in organogenesis: making the difference. , 2015, Trends in genetics : TIG.

[29]  H. Jang,et al.  Molecular Analysis of Alternative Transcripts of the Equine Cordon-Bleu WH2 Repeat Protein-Like 1 (COBLL1) Gene , 2015, Asian-Australasian journal of animal sciences.

[30]  Evan Z. Macosko,et al.  Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets , 2015, Cell.

[31]  R. Sandberg,et al.  Single-Cell RNA-Seq Reveals Dynamic, Random Monoallelic Gene Expression in Mammalian Cells , 2014, Science.

[32]  A. Fatica,et al.  Long non-coding RNAs: new players in cell differentiation and development , 2013, Nature Reviews Genetics.

[33]  W. Schulz,et al.  Specific changes in the expression of imprinted genes in prostate cancer--implications for cancer progression and epigenetic regulation. , 2012, Asian journal of andrology.

[34]  H. Sasaki,et al.  Genomic imprinting and its relevance to congenital disease, infertility, molar pregnancy and induced pluripotent stem cell , 2012, Journal of Human Genetics.

[35]  Y. Tarutani,et al.  Monoallelic gene expression and its mechanisms. , 2011, Current opinion in plant biology.

[36]  J. Bageritz,et al.  Single-Cell RNA Sequencing with Drop-Seq. , 2019, Methods in molecular biology.

[37]  Claude-Alain H. Roten,et al.  Theoretical and practical advances in genome halving , 2004 .