© 2015 Nature America, Inc. All rights reserved. authors authors contributed equally to this work. Raleigh, North Carolina, USA (D.L.A.) and Department of Computer Science, University of California, Los Angeles, Los Angeles, California, USA (W.W.). Epidemiology and Karolinska Biostatistics, Institutet, Stockholm, Sweden. College Station, Texas, USA. University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 4 at Chapel Hill, Chapel Hill, North Carolina, USA. 1 F reciprocal include to experiment our designed we in Therefore, variation epigenetic or genetic requires that process a mice, heterozygous in transcription in imbalance allelic detect to used be can (ASE) expression of allele-specific Examination alleles. parental two the of one either to transcripts assign to capacity the enhancing concurrently while variation genetic of study level The the mice. maximized design laboratory in tissues multiple in on expression gene origin parental and variation genetic of effects the investigated traits biomedical and diseases human with associated strongly nonetheless are structure, protein change to predicted not although that, SNPs of thousands of identification the through and populations and taxa divergent comparing studies recent in ent The appar sequences. increasingly become has sequence regulatory regulatory of or importance RNA protein-coding, in to assigned variation be can variation phenotypic most of basis genetic The complex imbalance cis complex tissues aid regulatory Complex Patrick F Sullivan David W Threadgill Wei Wang J Randal Nonneman Nashiya N Robinson Ryan J Buus Jeremy R Wang James J Crowley imbalance allelic pervasive identifies crosses mouse divergent highly in expression gene of allele-specific Analyses Nature Ge Nature Received 17 September 2014; accepted 26 January 2015; published online 2 March 2015; Lineberger Comprehensive Lineberger Cancer Comprehensive Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

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© 2015 Nature America, Inc. All rights reserved. somatic tissues are more abundant on the X chromosome than on on autosomes than chromosome X the on abundant more are tissues somatic considered are sion expres low with genes whether been has studies across results rate studies recent multiple tissues human and mouse cies spe eutherian several across studies microarray three by supported (Ohno’sthe autosomes hypothesis) on expression to equivalent roughly is expression X-chromosome of expressed are biallelically in the human scenario where ~15% of females, genes X-chromosome identified been viously pre have which of all tested, be could that genes the of 1.1% just for is rare for genes to escape X inactivation in mouse, with this occurring it that we confirm Furthermore, samples. tissue primary in evidence lines cell using before demonstrated been has this Although expression. similar have females and males some. First, for most of the genes on the X chromosome, we found that imprinting of studies controversial previous 2 in found genes of number large surprisingly the explain and may also allele paternal the of overexpression modest show 54) of (37 here described genes may partly explain why the majority of the newly identified imprinted the global allelic imbalance in favor of expression of the paternal allele Lastly, methylation. overall on effect minor overall an have only will cis strong create to likely are type later the of genes of promoters the in mutations that fact by the explained be could effect parent-of-origin this by not or affected are that genes between effects strain of size in selection to create classical imprinting. We propose that the natural difference by exploited been have may TSS the to close islands CpG at In imprinting. in other methylation words, parental differences small for imbalance. allelic responsible global mechanism the likely is development early during marks methylation of resetting origin–dependent of parent ferential CpG methylated differentially islands ( with associated are allele paternal ( TSSs genes their inconsistent to relative near effects strain smaller islands show to tend and CpG for enriched strongly are sexes and both crosses 3 all in allele paternal the of overexpression consistent have that genes 467 The crosses. reciprocal three in all and is present autosomes the all across distributed genes many affects This allele. paternal the of expression favored with brain, mouse in asymmetric be to appear expression gene on effects consensus. historical parent-of-origin However, the with line in is brain mouse in genes of imprinted number the that indicate results our then parent, one ing favor expression in imbalance allelic significant show that genes to genes subject to imprinting. If the definition of imprinting is restricted year. each created likely are variants regulatory proportion and the size of the human population, million new several this Given mutation. regulatory new a have should offspring 10 in 1 and mice average ~100 as human at Furthermore, gene a expression maintain level. constant to pressure selective under likely are study) this in (~15% variation regulatory without genes testable of proportion small the and tions, contribute to the broad phenotypic distributions seen in those popula Nature Ge Nature chromosome X the on genes all considers analysis Our ratios. sion Fig. Fig. We verified two forms of dosage compensation on the X chromo X the on compensation dosage of forms two We verified classical to ancestral is imbalance global this that Wehypothesize There have been conflicting reports regarding the number of mouse regulatory variants. On the other hand, mutations in CpG islands in CpG islands mutations hand, other On the variants. regulatory 40 , 6 47 ). In addition, genes with consistent overexpression of the the of overexpression consistent with genes addition, In ). , Supplementary Supplementary Fig. 10 4 8 but then contradicted in 2010 by an RNA-seq analysis of of analysis RNA-seq an by 2010 in contradicted then but 5 n 0 , their inclusion can lower median X:autosome expres X:autosome median lower can inclusion their , etics

57 ADVANCE ONLINE PUBLICATION ONLINE ADVANCE , 5 8 42– 50– de de novo . Because genes with no or low expression in in expression low or no with genes Because . 45 4 5 4 4 6 , 4 . This finding stands in sharp contrast to contrast sharp in stands finding This . 9 . The main factor contributing to dispa to contributing factor main The . 6 , and this controversy remains, despite despite remains, controversy this and , . Second, . we Second, found that the level overall mutations per generation ). ). These observations suggest that dif 3 1 40 . . Ohno’s was initially hypothesis , 4 1 , here we provide additional additional provide we here ,

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fied by creating a union of the databases from the following websites: by websites: a fied from the creating following of union the databases identi were imprinting of evidence previous with one2one.”Genes compara/homology ( Ensembl from identified org/phenotyp from acquired were phenotypes mouse at GeneScissors access (pseudogenes), misalignment read RNA-seq spurious of at correction found be can skewing h X-inactivation in factor to and ASReC com/p/suspenders com/p/lapels Scripts are provided to construct pseudogenomes ( URLs. humans. in traits genetic complex underlying mechanisms powe a vide variation regulatory incorporating to models Mouse subject are genes of majority vast the pressure. evolutionary under is sion that of the level gene expres strong evidence provides compensation and clearly supports Ohno’s hypothesis in mouse. This form of dosage 2. 1. reprints/index.ht at online available is information permissions and Reprints The authors declare no competing interests.financial pseudogenome construction and RNA-seq read alignment. S.H., I.K.P., J.R.W., C.E.W., C.-P.F., Z.Z., J.H., Z.G. and L.M. contributed to J.X., J.P.D., A.P.M. and D.L.A. contributed to data analysis and interpretation. statistical models and conducted analyses. W.V., A.B.L., D.W.T., L.M.T., K.K., prepared samples for expression profiling. W.S., F.Z., V.Z., Y.K. and W.W. developed C.R.Q. and Y.X. bred the mice and tissues. collected J.D.C., C.J.B., Z.Y. and T.J.G. edited it. D.R.M., G.D.S., T.A.B., R.J.B., M.E.C., S.D.H., J.S.S.,N.N.R., R.J.N., managed the project. J.J.C. and F.P.-M.d.V. drafted the manuscript, and all authors F.P.-M.d.V., J.J.C., L.M., F.Z., W.S., V.Z. and P.F.S. the designed study, and J.J.C. the National Institute of Mental Health. Medical General and Sciences (principal K01MH094406 investigator J.J.C.) from grants (principal R01GM074175 investigator F.Z.) from the National Institute of co-principal investigators F.P.-M.d.V. and P.F.S.). This work was also supported by of Excellence for Genome grants Sciences and(P50MH090338 P50HG006582, Institute of Mental Health/National Human Genome Research Institute Center M. Calabrese for helpful discussions. Major funding was provided by National We thank P. Mieczkowski, A. Brandt, E. Malc, M. Vernon, J. Brennan and online version of the pape Note: Any Supplementary Information and Source Data files are available in the accession under (GEO) Omnibus Expression codes. Accession the pape the of in version available are references associated any and Methods M mous http:// C AUTH A ttp://www.bios.unc.e ckn O

ethods In summary, our study demonstrates that in the laboratory mouse mouse laboratory in the that demonstrates In our study summary, MPETING FINANCIAL INTERESTS FINANCIAL MPETING ig MC & isn AC Eouin t w lvl i hmn ad chimpanzees. and humans in levels two at Evolution A.C. Wilson, & M.C. King, the in elements DNA of encyclopedia integrated An Consortium. Project ENCODE Science genome. human ebook.org/catalog.ph o O www.geneimprint.com Expression data can be viewed at data can be viewed Expression wledgments R R C

188 O r NTRIBUTI ful complement to null mutants null to complement ful / , 107–116 (1975). 107–116 , m es.shtm ) and perform diploid alignment ( alignment diploid perform and ) l . Nature Expression data can be acquired from the Gene Gene the from acquired be can data Expression / r ). An R package for jointly analyzing TReC and and TReC analyzing jointly for package R An ). . _method.htm r . l . . Orthologous genes for human and mouse were

O du/~feizou/software/ 489 http://csbio.u NS , 57–74 (2012). 57–74 , p?catalog=imprintin http://www. / , http://igc.otago.ac l uig h ctgr “ortholog_ category the using ) nc.edu/genescissors http://csbio.unc.ed http://www.informat ensembl.org/info/gen cis rxSe 1 GSE4455 regulatory variation. variation. regulatory 9 in the search for the the for search the in g .nz q . http://code.google http://www.nature.c . For detection and . For detection s e l c i t r A http://code / 20 and , 2 1 5 should pro should . / http://www. . Knockout . Knockout u/gecco

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© 2015 Nature America, Inc. All rights reserved. cate the numbers of maternal and paternal alleles (SNPs and indels) observed (SNPs observed and indels) alleles and of paternal cate numbers the maternal indi to read aligned each annotated we Third, mm9). to relative coordinates pseudogenome the alter indels as necessary was (which read aligned each of the strings CIGAR and rewriting positions by dinates the alignment updating to reads mm9 coor from the we pseudogenome-aligned coordinates mapped Second, release). 2011 (June and WSB/EiJ PWK/PhJ CAST/EiJ, included that effort sequencing large-scale a by reported variants all We(mm9). included structed by incorporating all known SNPs and indels into the C57BL/6 genome con were approximations Pseudogenomes of and indel 3 a bases). maximum read 100-bp per allowed two mismatches bp, two 25 segment, per of allowed mismatches length segment including parameters default (v1.4; TopHat appropriate the to aligned (ref. were ­‘pseudogenomes’ diallel) the in cells nine the of (six hybrid F each from reads First, sequence. reference the and genomes between parental the distance genetic in differences by caused bias minimize to and ity qual alignment improve to sequence reference alignment the into variants genetic approach has the advantage all of known strain-specific incorporating diversity genetic considerable containing line for subspecies mouse alignment. RNA-seq: barcodes of lanes. sequencing assignments to or and of indicators group combination these between each for associations 2) sex. × and 3 strain × maternal (3 strain, groups paternal 18 into partitioned be can was portion each randomly and assigned tosequence portions, one four and lane into of density onedivided was machine. cluster sample The 384 each in portions quality, (4 effects × 96 reads. samples) machine and paired-end lane 100-bp for generate account Toto used was 2000 HiSeq Illumina The pools. equimolar a of multiplexed total eight at yielding sequencing, before using concentrations pooled were samples quantified were selected randomly 12 Libraries and barcodes. fluorometry, different two least at assigned Sample RNA was sample each and TruSeq input, as used was sample RNA per total of microgram Illumina One the (AD001–AD012). adaptors using indexed unique 12 with v2 Kit preparation Preparation library for 48 of preparation. sample RNA-seq: Technologies). Agilent (Bioanalyzer, verified platform microfluidics a was using quality RNA and Technologies), Life Fluorometer, 2.0 (Qubit RNA Total Kit, Promega). Purification RNA LEV concentration was by measured fluorometry Tissue 16 (Maxwell instrumentation automated using sue extraction. RNA Products). (BioSpec unit BioPulverizer a using pulverized and nitrogen liquid in frozen snap dissected, rapidly were lobes) (all and lungs (both) kidneys lobe), (left liver brain, Whole cage. home were euthanized between 10 and 12 a.m., immediately after removal from their without anesthesia (to avoid confounding effects on gene expression). All mice collection. Tissue enrichment. for cage each in present were material available were RMH3000 Prolab Purina Waterand bedding. Bed-O-Cob laboratory-grade with cages humidity. Mice were housed in standard 20 cm × 30 cm relative ventilated polysulfone 40–50% with °C 20–24 at maintained was room housing The a.m. 6 at on turned lights with schedule dark 10-h light, 14-h a Mice on maintained were Laboratory. Jackson the from acquired founders from removed tions genera six than fewer were that mice from Carolina North of University the and WSB/EiJ. CAST/EiJ, PWK/PhJ strains were mice All bred at wild-derived Mice. of University the of Committee by Use Carolina. and approved North and Care 1996) Animal Council, Institutional the Research National Resources, Animal Animals Laboratory of Use and Care the for Guide statement. Ethical ONLINE doi: 10.1038/ng.3222 The mice used in this study were inbred and reciprocal F

METHODS Total RNA was extracted from ~25 mg of powdered tis powdered of mg ~25 from extracted was RNA Total Mice were killed at 23 23 at killed were Mice All mouse work was conducted in compliance with the the with compliance in conducted was work mouse All We developed a customized RNA-seq alignment pipe RNA-seq alignment Wea customized developed 2 2 ), representing each of the parental genomes using using genomes parental the of each representing ), ad libitum ad The 96 samples were randomized to batches to batches randomized were samples 96 The . A small section of PVC pipe and nestlet nestlet and of PVC pipe section . A small ± χ 1 d of age by cervical dislocation dislocation cervical by age of d 1 2 tests confirmed no significant significant no confirmed tests (Institute of Laboratory Laboratory of (Institute 1 hybrids of the 22– 2 4 . . This 4 1 ------

isoform/countReads. Exon position information was assigned on the basis of basis on the assigned was information position Exon isoform/countReads. function R the using gene a of regions exonic the overlapped that reads end We other). the to from of a mapped of number the reads as gene number the paired- counted then were none and parent one (all from consistent were fully reported was it alleles if only parent a to assigned was it indel, or SNP and paternal the between maternal Ifstrains. a read paired-end overlapped more than one heterozygous heterozygous was that overlapped indel or end SNP either one if least at specific allele was read paired-end A ASReC). or paired-end reads (only for allele-specific F maternal and paternal of numbers the and TReC) or count, read total mice; F and inbred both (for reads paired-end of number total the RNA-seq: with RNA-seq: read assignment. control. quality passed samples strain for the sex chromosomes, autosomes and mitochondrial genome. Ninety for on the of proportions expectations reads that to should align each parental stages. annotation and remapping same by the followed necessary, was alignment pseudogenome single a only mice, inbred For file. alignment single a into combined were files alignment resulting the and sample, a of lanes the all to separately applied was step final This once). combined were alignments such that a to read was the aligning two in same counted position both alignments the (i.e., alignments separate the of union to alignments Finally, were maternal and merged pseudogenomes paternal by computing the proper ASE. determining reads paired-end more of use the in a given read and its paired-end mate. Considering paired-end mates allowed indicating multiple 96-well plates and “dissection” is a categorical variable variable categorical a is “dissection” and plates 96-well multiple indicating F reciprocal gin” is a vector for comparisons of reciprocal F where “strain” is a vector for comparisons of two inbred strains, “parent of ori as each effects follows: for sex and models dominance parent-of-origin, fixed-effect strain, for linear test to fitted transcript we strains, inbred the between analysis. statistical Microarray: below). provided are details (full for effects sex and dominance models parent-of-origin, strain, for test to fixed-effect transcript each linear fitted we strains, inbred the F between reciprocal crosses and strains and inbred For function (PCA). link analysis average the principal-component with hclust function R the hierarchical using applied we clustering arrays, the of performance overall the evaluate To annotation. mRNA without those and sets probe control excluding after sets strains inbred mouse 3 the in SNPs known any ing transcripts. During of normalization, we masked 78,632 probes levels (~10% of all probes) contain expression normalized the estimate to normalization) gene sketch-quantile and Affymetrix polish (median the settings default in with console expression implemented (RMA) method average robust multiarray the used We protocols. manufacturer’s the to according Affymetrix Affymetrix to from Mouse 1.1 Plate ST Gene instrument a using arrays 96-Array GeneTitan hybridized was RNA-seq for used mice same the from RNA control. quality and processing Microarray: al. et analysis. RNA-seq: statistical covariate. a as sample each for reads of number total the We included samples. across gene that of expression the of and were was gene thus length gene analyses constant specific in comparisons all because length gene for correct to need no was There 2012). February 14 transcriptome annotations from Ensembl (Release 66, based on mm9; accessed After After alignment, we performed a series of quality control checks capitalizing y 2 4 + = as well as in the the in as well as + × + + b b 1 0 b b b 9 8 5 1 st plat crosses, “sex” indicates female, “plate” is a categorical variable variable categorical a is “plate” female, indicates “sex” crosses, rain st d e rain sse + p x Supplementary Note Supplementary b + isse 2 b parent 6 Three counts were obtained for each gene assessed c ttion Statistical analysis is described in detail in in Zou detail is described analysis Statistical aren For inbred strains and reciprocal F reciprocal and strains inbred For + of o t e origin o f rigi 1 hybrids; allele-specific read count, hybrids; allele-specific + s n . 1 b × crosses, “dominance” indicates Brain, liver, kidney and lung lung and kidney liver, Brain, 3 ex dominanc + 4 . We used 28,310 probe probe 28,310 Weused . b 7 Nature Ge Nature dominanc s e + b 4 ex s e × 1 n crosses crosses ex etics 1 1 - -

© 2015 Nature America, Inc. All rights reserved. we randomly sampled 467 and 116 genes from the whole gene list and calculated from CpG islands ( away further be to tended genes overexpressed maternally and genes, sistent Paternally overexpressed genes tended to be closer to CpG islands than incon distributions. respective generate to expression) parental inconsistent (with genes expressed of remainder the for those to distances these Wecompared overexpressed. maternally consistently were that genes 116 all and expressed the nearest CpG island for all 467 genes that were consistently over paternally expected. than higher was proportion observed the autosomes, 19 of 15 for that, Wefound chromosome. corresponding the for genes overexpressed paternally of tion with null: the under proportions expected be would what chromosome-wise these compared we Then sexes. both and crosses three from results pooling after chromosome each within genes We effect. of of the proportion such recorded parent-of-origin same direction the had genes neighboring whether checked we sex, each and cross each For in shown clustering of the magnitude the Toquantify clustering. of evidence was there and autosome, one into collapsed then and case) each in significant were (and and sex of cross combination for each result separately tests median These were from simulations). 2,000 performed the (we used test a using Wilcoxon 50% rank-sum from different was portion subset of 1,000 genes, we tested whether the paternal expression expected pro (this test is therefore conservative yet still highly Forsignificant). each random of avoid to subset genes random a used We times. 2,000 genes) imprinted known (minus in shown bias sion data. RNA-seq in bias expression Paternal and (FDR) rate discovery the if false significant be to tests declared used we correction, multiple-testing For follows: as effects sex and dominance parent-of-origin, strain, the Wetested dates. dissection different indicating Nature Ge Nature Se St Dominanc e Parent-of-origin To further explore this clustering, we calculated the distance from the TSS to every on observed was imbalance allelic on effect parent-of-origin This rain e x ffect effect : : e e H n : : 4 0 H ffect etics P b b 1 0 -value inflation due to possible correlation between genes genes between correlation possible to due inflation -value b b : : f Fig. Fig. 6 = = ffect H Figure 6 Figure = 3 0 6 5 : : b b P H 1 5 c value using Fisher’s combined probability test. probability Fisher’s using combined value ). ). To of test formally the this difference, significance b b 2 0 0 = Figure 6 Figure = = b b a = = , we permuted a random subset of 1,000 genes genes 1,000 of subset random a permuted we , vs 7 = = : . 1 7 H v 0 1 6 b v 0 , we performed the following procedure. procedure. following the , we performed q s. b b value was <0.05. was value 5 1 s. v 0 H ≠ H 3 1 p o 0 s. 0 : 2 b b + (1 − − (1 + 0 : H To quantify the paternal expres paternal the To quantify b b 0 r 5 4 ≠ ≠ : : b b ≠ ≠ 6 2 ≠ or ≠ ≠ p or ) o 0 2 7 , where where , 0 r ≠ ≠ 0 0 0 b b p 7 6 is the propor the is or ≠ 0 0 - - - - -

we examined distributions for all combinations of methylation group (mater of for methylation combinations all distributions we examined In words, to other overexpressed. parental with respect tion of distances these island for each parentally biased methylation group and examined the distribu group. reference a as used were methylation preferential no with islands CpG remaining The methylated. paternally preferentially be to island CpG than we higher the proportion proportion, maternal methylation the declared a had paternal mice if both Likewise, analysis. of this purposes for the ylated, we proportion, this CpG declared island meth to maternally be preferentially paternal the than higher proportion methylation a had maternal mice if both counts over methylation each CpG island and applied a criterion: simple filter this CpG integrated we cross, first just reciprocal one per mouse included set data Because mice. Cast/EiJ and 129X1/SvJ of hybrids reciprocal from data DNA methylation brain parent-of-origin whole-genome of consists set Xie data by publication recent a from Omnibus) Expression Gene methylation ( in bias parent-of-origin with islands CpG to closer were allele paternal the from overexpression consistent methylation. with DNA genes and whether bias Wetested expression paternal between Relationship the as 10 × 1 and result) extreme empirical as was none permutations, 100,000 of (out genes expressed resulting The data. unperturbed squared from deviation randomly sampled genes was larger than the one from the calculated and times the mean squared of deviation Wethe curves. repeated this procedure 100,000 CpG islands was itself also significant ( significant also itself was islands CpG ( (permutation islands CpG methylated maternally for enrichment greatest the had genes expressed to genes of class each inconsistent CpG Weisland. over found that paternally than closer be to tended genes overexpressed paternally consistent whether island (bp)) is shown in CpG to nearest TSS expressed: (bp)/inconsistently CpG island to nearest TSS log ratio: log following the inconsistently using and genes, genes expressed overexpressed paternally consistently of parison com A island. CpG closest the and TSS the between distance median the of permutations 10,000 used we robust more result the make to and group, est group, we calculated distance to a down-sampled subset equivalent to per count the island CpG small differential to To due bias total. avoid in combinations six and and group and nal, paternal other) maternal), (paternal overexpressed P Supplementary Fig. 10 Fig. Supplementary = 0.0034). This greater enrichment for maternal over paternal methylated methylated paternal over maternal for enrichment greater This 0.0034). = Next, we calculated the distance from each gene’s TSS to the closest CpG CpG closest the to gene’s TSS each from distance the calculated we Next, P = 0), followed by paternally methylated CpG islands islands CpG methylated paternally by followed 0), = Supplementary Supplementary Figure 10 ). To accomplish this, we used a data set ( a set data we ). To used this, accomplish P value as the proportion of times where the mean mean the where times of proportion the as value −5 for maternally overexpressed genes. overexpressed maternally for P values were <1 × 10 × <1 were values P = 0.0015). = . . In short, this plot examines 10 (paternally expressed: expressed: (paternally −5 for paternally over paternally for doi: 10.1038/ng.3222 et al. et GSE3372 2 8 . This This . 2 ------,