Decoding neural transcriptomes and epigenomes via high-throughput sequencing

The mammalian brain is an evolutionary marvel in which engraving and re-engraving of cellular states enable complex information processing and lifelong maintenance. Understanding the mechanisms by which neurons alter and maintain their molecular signatures during information processing is a fundamental goal of neuroscience. Next-generation sequencing (NGS) technology is rapidly transforming the ability to probe the molecular basis of neuronal function. NGS can define not only the complete molecular signatures of cells by transcriptome analyses but also the cascade of events that induce or maintain such signatures by epigenetic analyses. Here we offer some general and practical information to demystify NGS technology and highlight its potential to the neuroscience field. We start with discussion of the complexity of the nervous system, then introduce various applications of NGS with practical considerations and describe basic principles underlying various NGS technologies. Finally, we discuss emerging NGS-related technologies for the neuroscience field.

[1]  C. Nusbaum,et al.  Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements. , 2006, Genome research.

[2]  Zachary D. Smith,et al.  Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling , 2012, Genome Biology.

[3]  G. Ming,et al.  Neuronal Activity–Induced Gadd45b Promotes Epigenetic DNA Demethylation and Adult Neurogenesis , 2009, Science.

[4]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[5]  F. Tang,et al.  Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing , 2013, Genome research.

[6]  S. Henikoff,et al.  A simple method for gene expression and chromatin profiling of individual cell types within a tissue. , 2010, Developmental cell.

[7]  S. Kingsmore,et al.  Comprehensive human genome amplification using multiple displacement amplification , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Neil L Kelleher,et al.  Pervasive combinatorial modification of histone H3 in human cells , 2007, Nature Methods.

[9]  R. Paro,et al.  Analysis of chromatin structure by in vivo formaldehyde cross-linking. , 1997, Methods.

[10]  William Stafford Noble,et al.  Global mapping of protein-DNA interactions in vivo by digital genomic footprinting , 2009, Nature Methods.

[11]  S. Batalov,et al.  Antisense Transcription in the Mammalian Transcriptome , 2005, Science.

[12]  J. Sweatt,et al.  DNA methylation and memory formation , 2010, Nature Neuroscience.

[13]  V. Iyer,et al.  FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin. , 2007, Genome research.

[14]  S. Turner,et al.  Real-time DNA sequencing from single polymerase molecules. , 2010, Methods in enzymology.

[15]  Christopher J. Lee,et al.  Alternative splicing in the nervous system: an emerging source of diversity and regulation , 2003, Biological Psychiatry.

[16]  K. Malinovskaja,et al.  DNA Methylation Regulates Cocaine-Induced Behavioral Sensitization in Mice , 2010, Neuropsychopharmacology.

[17]  Rona S. Gertner,et al.  Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells , 2013, Nature.

[18]  Nicholas T. Ingolia,et al.  Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes , 2011, Cell.

[19]  M. Selbach,et al.  Global quantification of mammalian gene expression control , 2011, Nature.

[20]  Alexander Varshavsky,et al.  Mapping proteinDNA interactions in vivo with formaldehyde: Evidence that histone H4 is retained on a highly transcribed gene , 1988, Cell.

[21]  J. Weissman,et al.  Nascent transcript sequencing visualizes transcription at nucleotide resolution , 2011, Nature.

[22]  M. Aladjem,et al.  DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells , 2006, PLoS genetics.

[23]  G. Hon,et al.  Base-Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome , 2012, Cell.

[24]  Nathan C. Sheffield,et al.  The accessible chromatin landscape of the human genome , 2012, Nature.

[25]  M. Groudine,et al.  Chromosomal subunits in active genes have an altered conformation. , 1976, Science.

[26]  J. Sweatt,et al.  Epigenetic Regulation of bdnf Gene Transcription in the Consolidation of Fear Memory , 2008, The Journal of Neuroscience.

[27]  Neil R Smalheiser,et al.  Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training. , 2011, RNA.

[28]  S. Sunkin,et al.  Specific expression of long noncoding RNAs in the mouse brain , 2008, Proceedings of the National Academy of Sciences.

[29]  Dustin E. Schones,et al.  High-Resolution Profiling of Histone Methylations in the Human Genome , 2007, Cell.

[30]  Chuan He,et al.  Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine , 2011, Science.

[31]  Benjamin A. Garcia,et al.  Asymmetrically Modified Nucleosomes , 2012, Cell.

[32]  Zachary D. Smith,et al.  Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling , 2011, Nature Protocols.

[33]  D. Branton,et al.  The potential and challenges of nanopore sequencing , 2008, Nature Biotechnology.

[34]  Guoping Fan,et al.  Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain , 2013, Nature Neuroscience.

[35]  James Allan,et al.  Micrococcal Nuclease Does Not Substantially Bias Nucleosome Mapping , 2012, Journal of molecular biology.

[36]  F. Crick Neurobiology: Memory and molecular turnover , 1984, Nature.

[37]  ENCODEConsortium,et al.  An Integrated Encyclopedia of DNA Elements in the Human Genome , 2012, Nature.

[38]  D. Galas,et al.  DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. , 1978, Nucleic acids research.

[39]  David G. Knowles,et al.  Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs , 2012, Genome research.

[40]  Cole Trapnell,et al.  Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. , 2010, Nature biotechnology.

[41]  Kamel Jabbari,et al.  Cytosine methylation and CpG, TpG (CpA) and TpA frequencies. , 2004, Gene.

[42]  Z. Weng,et al.  A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome , 2006, Cell.

[43]  Yi Zhang,et al.  Dnmt3a-Dependent Nonpromoter DNA Methylation Facilitates Transcription of Neurogenic Genes , 2010, Science.

[44]  M. Kupiec,et al.  Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq , 2012, Nature.

[45]  L. Blanco,et al.  Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[46]  J. Dekker,et al.  Capturing Chromosome Conformation , 2002, Science.

[47]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[48]  David R. Liu,et al.  Conversion of 5-Methylcytosine to 5- Hydroxymethylcytosine in Mammalian DNA by the MLL Partner TET1 , 2009 .

[49]  E. Marcotte,et al.  Insights into the regulation of protein abundance from proteomic and transcriptomic analyses , 2012, Nature Reviews Genetics.

[50]  Erin M. Wissink,et al.  Rapid activity-induced transcription of Arc and other IEGs relies on poised RNA polymerase II , 2011, Nature Neuroscience.

[51]  G. Hon,et al.  Adult tissue methylomes harbor epigenetic memory at embryonic enhancers , 2013, Nature Genetics.

[52]  R. Sandberg,et al.  Full-Length mRNA-Seq from single cell levels of RNA and individual circulating tumor cells , 2012, Nature Biotechnology.

[53]  Bing Ren,et al.  Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine , 2012, Nature Protocols.

[54]  J. Morrison,et al.  Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens , 2010, Nature Neuroscience.

[55]  E. Nestler,et al.  Epigenetic mechanisms of depression and antidepressant action. , 2013, Annual review of pharmacology and toxicology.

[56]  A. Gingras,et al.  Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family , 2012, Cell.

[57]  Leighton J. Core,et al.  Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters , 2008, Science.

[58]  B. Steensel,et al.  Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture–on-chip (4C) , 2006, Nature Genetics.

[59]  Lee E. Edsall,et al.  Human DNA methylomes at base resolution show widespread epigenomic differences , 2009, Nature.

[60]  B. S. Manjunath,et al.  Identification of piRNAs in the central nervous system. , 2011, RNA.

[61]  A. Maiti,et al.  Thymine DNA Glycosylase Can Rapidly Excise 5-Formylcytosine and 5-Carboxylcytosine , 2011, The Journal of Biological Chemistry.

[62]  L. E. McDonald,et al.  A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Christian Panse,et al.  Identification of Combinatorial Patterns of Post-Translational Modifications on Individual Histones in the Mouse Brain , 2012, PloS one.

[64]  S. Henikoff,et al.  Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. , 2012, Genome research.

[65]  M. Stephens,et al.  RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. , 2008, Genome research.

[66]  J. Kjems,et al.  Natural RNA circles function as efficient microRNA sponges , 2013, Nature.

[67]  M. Cynader,et al.  Distinct DNA methylation patterns of cognitive impairment and trisomy 21 in down syndrome , 2013, BMC Medical Genomics.

[68]  M. Robinson,et al.  Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA , 2012, Genome research.

[69]  C. Wahlestedt,et al.  Inhibition of natural antisense transcripts in vivo results in gene-specific transcriptional upregulation , 2012, Nature Biotechnology.

[70]  C. Allis,et al.  Translating the Histone Code , 2001, Science.

[71]  R. Palmiter,et al.  Cell-type-specific isolation of ribosome-associated mRNA from complex tissues , 2009, Proceedings of the National Academy of Sciences.

[72]  H. Hayatsu,et al.  Formation of Diastereomers of 5, 6-Dihydrothymine-6-sulfonate by Deamination of 5-Methylcytosine with Bisulfite , 1975 .

[73]  William Stafford Noble,et al.  Sequence and chromatin determinants of cell-type–specific transcription factor binding , 2012, Genome research.

[74]  Yang Wang,et al.  Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA , 2011, Science.

[75]  F. Crick Memory and molecular turnover. , 1984, Nature.

[76]  A. Bird DNA methylation patterns and epigenetic memory. , 2002, Genes & development.

[77]  Vijay K. Tiwari,et al.  DNA-binding factors shape the mouse methylome at distal regulatory regions , 2011, Nature.

[78]  Nancy F. Hansen,et al.  Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry , 2008, Nature.

[79]  Andres Metspalu,et al.  In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes , 2013, Nucleic acids research.

[80]  Alan P. Wolffe,et al.  Disruption of Higher-Order Folding by Core Histone Acetylation Dramatically Enhances Transcription of Nucleosomal Arrays by RNA Polymerase III , 1998, Molecular and Cellular Biology.

[81]  Robert Shapiro,et al.  Reactions of Uracil and Cytosine Derivatives with Sodium Bisulfite , 1970 .

[82]  R. Flavell,et al.  Interchromosomal associations between alternatively expressed loci , 2005, Nature.

[83]  George M. Church,et al.  Highly Multiplexed Subcellular RNA Sequencing in Situ , 2014, Science.

[84]  Zhike Lu,et al.  Identification of 67 Histone Marks and Histone Lysine Crotonylation as a New Type of Histone Modification , 2011, Cell.

[85]  A. Graessmann,et al.  Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[86]  J. Issa,et al.  DNA methylation does not stably lock gene expression but instead serves as a molecular mark for gene silencing memory. , 2012, Cancer research.

[87]  S. Haggarty,et al.  HDAC2 negatively regulates memory formation and synaptic plasticity , 2009, Nature.

[88]  Colin A. Johnson,et al.  Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex , 1998, Nature.

[89]  Steven Henikoff,et al.  High-resolution mapping of transcription factor binding sites on native chromatin , 2013, Epigenetics & Chromatin.

[90]  A. Bird,et al.  Genomic DNA methylation: the mark and its mediators. , 2006, Trends in biochemical sciences.

[91]  G. Ming,et al.  Hydroxylation of 5-Methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain , 2011, Cell.

[92]  S. Horvath,et al.  Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing , 2013, Nature.

[93]  E. Birney,et al.  High-resolution genome-wide in vivo footprinting of diverse transcription factors in human cells. , 2011, Genome research.

[94]  G. Ming,et al.  A unifying hypothesis on mammalian neural stem cell properties in the adult hippocampus , 2012, Current Opinion in Neurobiology.

[95]  David J. Arenillas,et al.  JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles , 2013, Nucleic Acids Res..

[96]  L. Wessels,et al.  Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions , 2008, Nature.

[97]  Steven Henikoff,et al.  Surveying the epigenomic landscape, one base at a time , 2012, Genome Biology.

[98]  J. Sweatt,et al.  Cortical DNA methylation maintains remote memory , 2010, Nature Neuroscience.

[99]  Li-Huei Tsai,et al.  Recovery of learning and memory is associated with chromatin remodelling , 2007, Nature.

[100]  D. Bodian A SUGGESTIVE RELATIONSHIP OF NERVE CELL RNA WITH SPECIFIC SYNAPTIC SITES. , 1965, Proceedings of the National Academy of Sciences of the United States of America.

[101]  P. Greengard,et al.  A Translational Profiling Approach for the Molecular Characterization of CNS Cell Types , 2008, Cell.

[102]  N. Friedman,et al.  Comprehensive comparative analysis of strand-specific RNA sequencing methods , 2010, Nature Methods.

[103]  A. Bird,et al.  Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[104]  P. Bickel,et al.  System wide analyses have underestimated protein abundances and the importance of transcription in mammals , 2012, PeerJ.

[105]  Jessica L. Crisci,et al.  Human-Specific Histone Methylation Signatures at Transcription Start Sites in Prefrontal Neurons , 2012, PLoS biology.

[106]  D. Botstein,et al.  Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF , 2001, Nature.

[107]  Nathan C. Sheffield,et al.  Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. , 2011, Genome research.

[108]  K. Morris,et al.  Bidirectional Transcription Directs Both Transcriptional Gene Activation and Suppression in Human Cells , 2008, PLoS genetics.

[109]  A. Gavin,et al.  Cell type–specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP , 2012, Nature Protocols.

[110]  Matthew D. Schultz,et al.  Global Epigenomic Reconfiguration During Mammalian Brain Development , 2013, Science.

[111]  Rodolfo R. Llinás,et al.  The contribution of Santiago Ramon y Cajal to functional neuroscience , 2003, Nature Reviews Neuroscience.

[112]  A. Feinberg,et al.  Genome-wide methylation analysis of human colon cancer reveals similar hypo- and hypermethylation at conserved tissue-specific CpG island shores , 2008, Nature Genetics.

[113]  David R. Liu,et al.  The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing , 2010, PloS one.

[114]  A. Tanay,et al.  Single cell Hi-C reveals cell-to-cell variability in chromosome structure , 2013, Nature.

[115]  M. Vermeulen,et al.  Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. , 2014, Nature chemical biology.

[116]  F. Gage,et al.  Epigenetic choreographers of neurogenesis in the adult mammalian brain , 2010, Nature Neuroscience.

[117]  Data production leads,et al.  An integrated encyclopedia of DNA elements in the human genome , 2012 .

[118]  F. Gage,et al.  RNA-sequencing from single nuclei , 2013, Proceedings of the National Academy of Sciences.

[119]  M. Fedurco,et al.  BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies , 2006, Nucleic acids research.

[120]  T. Dallman,et al.  Performance comparison of benchtop high-throughput sequencing platforms , 2012, Nature Biotechnology.

[121]  Catalin C. Barbacioru,et al.  mRNA-Seq whole-transcriptome analysis of a single cell , 2009, Nature Methods.

[122]  D. Zilberman,et al.  Genome-Wide Evolutionary Analysis of Eukaryotic DNA Methylation , 2010, Science.

[123]  J. Ausió,et al.  Modulation of Chromatin Folding by Histone Acetylation (*) , 1995, The Journal of Biological Chemistry.

[124]  Madeleine P. Ball,et al.  Neuronal activity modifies DNA methylation landscape in the adult brain , 2011, Nature Neuroscience.

[125]  I. Amit,et al.  Comprehensive mapping of long range interactions reveals folding principles of the human genome , 2011 .

[126]  Benjamin A Garcia,et al.  Analytical tools and current challenges in the modern era of neuroepigenomics , 2014, Nature Neuroscience.

[127]  Mark Akeson,et al.  Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands , 2013, Proceedings of the National Academy of Sciences.

[128]  T. Richmond,et al.  Crystal structure of the nucleosome core particle at 2.8 Å resolution , 1997, Nature.

[129]  Martin J Aryee,et al.  Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts , 2009, Nature Genetics.

[130]  Mazhar Adli,et al.  Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq , 2011, Nature Protocols.

[131]  A. Mortazavi,et al.  Genome-Wide Mapping of in Vivo Protein-DNA Interactions , 2007, Science.

[132]  B. Pugh,et al.  Comprehensive Genome-wide Protein-DNA Interactions Detected at Single-Nucleotide Resolution , 2011, Cell.

[133]  David G. Knowles,et al.  The GENCODE v7 catalog of human long noncoding RNAs: Analysis of their gene structure, evolution, and expression , 2012, Genome research.

[134]  P. Sorensen,et al.  The majority of total nuclear-encoded non-ribosomal RNA in a human cell is 'dark matter' un-annotated RNA , 2010, BMC Biology.

[135]  Han Xu,et al.  Analysis of optimized DNase-seq reveals intrinsic bias in transcription factor footprint identification , 2013, Nature methods.

[136]  Timothy J. Durham,et al.  "Systematic" , 1966, Comput. J..

[137]  N. Heintz,et al.  The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain , 2009, Science.

[138]  P. Jin,et al.  Genome-wide Profiling of 5-Formylcytosine Reveals Its Roles in Epigenetic Priming , 2013, Cell.

[139]  S. Haggarty,et al.  HDAC2 negatively regulates memory formation and synaptic plasticity , 2009, Nature.

[140]  Y. Wataya,et al.  Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. , 1970, Biochemistry.

[141]  Christoph Bock,et al.  Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk , 2012, Genome research.

[142]  S. Kouidou,et al.  Effect of chronic heroin and cocaine administration on global DNA methylation in brain and liver. , 2013, Toxicology letters.

[143]  Yi Zhang,et al.  Genome-wide Analysis Reveals TET- and TDG-Dependent 5-Methylcytosine Oxidation Dynamics , 2013, Cell.

[144]  Allen D. Delaney,et al.  Conserved Role of Intragenic DNA Methylation in Regulating Alternative Promoters , 2010, Nature.

[145]  J. Sweatt,et al.  Covalent Modification of DNA Regulates Memory Formation , 2008, Neuron.

[146]  Xavier Estivill,et al.  A myriad of miRNA variants in control and Huntington’s disease brain regions detected by massively parallel sequencing , 2010, Nucleic acids research.

[147]  Anton J. Enright,et al.  Materials and Methods Figs. S1 to S4 Tables S1 to S5 References and Notes Micrornas Regulate Brain Morphogenesis in Zebrafish , 2022 .

[148]  G. Kreiman,et al.  Widespread transcription at neuronal activity-regulated enhancers , 2010, Nature.

[149]  Nadav S. Bar,et al.  Landscape of transcription in human cells , 2012, Nature.

[150]  G. Ming,et al.  Genome-wide antagonism between 5-hydroxymethylcytosine and DNA methylation in the adult mouse brain , 2014, Frontiers in Biology.

[151]  Gene W. Yeo,et al.  An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells , 2009, Nature Structural &Molecular Biology.