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Nijmegen Centre for Molecular Life Sciences Molecular Biology Allele specific RNA-Seq The Case of X-inactivation Hendrik Marks August 25 th 2011 (Friday 14:00-14:45) NBIC Advanced RNA-seq course, Academic Medical Center, Amsterdam

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  • Nijmegen Centre for Molecular Life SciencesMolecular Biology

    Allele specific RNA-SeqThe Case of X-inactivation

    Hendrik MarksAugust 25th 2011

    (Friday 14:00-14:45)NBIC Advanced RNA-seq course, Academic Medical Center, Amsterdam

  • Karyotype diploid chromosomes; Gene expression from both alleles

    From each chromosome two copies, for each gene two copies

    What is the expression from each allele?

    XX

    Female mouse

    Microarray or regular RNA-Seq: average info for both alleles

  • Wang et al., 2008 (Plos One)

    Polymorphisms between both chromosomes:Allele specific assignment of reads over SNPs

    20*G and 16*T; Approximately equal for both alleles

  • Preferably in a defined setting (parental sequences known)

    A/T G/A C/G100/58 38/61 24/32

    Which of the nucleotides of the SNPs belong to each other

  • X CastM.m. castaneus

    129Inbred strainMix of M.m. DomesticusAnd M.m. musculus

    Either use model systems with known genomes, or sequence parents to work with a defined genome

    Cast129

    A/T G/A C/G100/58 38/61 24/32162/151

    Total

  • p(binomial) = probability that random events result in observed binary counts. e.g. probability of flipping coin 6 times and getting 1 or fewer heads (5 or more tails) is 10.9%

    Is a gene mono-allelicly expressed?

    Babak et al., 2008 (Current Biology)

    p=0.65p=0.11

  • A/T G/A C/G100/58 38/61 24/32162/151

    p=0.28 p=0.0005 p=0.006 p=0.17

    Various reports:- Per SNP per exon- Sum SNP counts per exon- Sum SNP counts per transcript

    To red! both to blue!

    Multiple SNPs over genes

  • - Statistics of allelic expression are dependent on tags counts, so dependent on the level of gene expression

    - Depending on the number of SNPs in transcripts

    - With current methods: Gradual scale of allele specific expression; when do youconsider a gene to be allele specific; what binomial p-value; what fold differencebetween both alleles?

    - Different SNPs in exons and different exons might point towards different directions; calculate per SNP, per exon, per transcript? Differences in numberswithin exons due to mapibility issues etc.

    - Difference between allelic expression (estimated at 20% of the genes, eg strainspecific expression) and imprinted genes (maternally or paternally expressed; onlya handful like H19, Cdknc and Igf2)

    Considerations for allele specific mappings

  • C57xCAST C=111, T=0 p=3.8e-34

    CASTxC57 C=2, T=34 p=9.7e-9

    Babak et al., 2008 (Current Biology)

    Example of an imprinted gene

  • The case of X inactivationStudying allele specific expression over timecourses

  • Kinetics of X-linked silencing in mouse ES cells using mRNA-Seq

    (Wutz and Gribnau, 2007; Curr Opin Genet Dev.)

    Illumina Hi-Seq

    Illumina GA IIx

    2i

    Undiff (pluripotentgroundstate) NPCs (Nestin stained)

    (Splinter et al., 2011; Genes Dev )

    EB formationtimecourse

    (From Web)

    (Picture from Rougeulle et al., 2004; Mol Cell Biol.)

  • X CastM.m. castaneus

    129Inbred strainMix of M.m. DomesticusAnd M.m. musculus

    XCI in polymorphic mouse ES cells

    X-chromosomes

    Xa (Cast)Xi (129)

    Cast129

    mRNA-Seq on polyA+ RNA

  • Massive allele specific mappings of the mRNA-seq

    GSNAP as preferred algorithm for mapping of the tags:1. Performs very well in general (next slide)2. Allows inclusions as well as de novo detection of splice junctions3. Allows SNP tolerant mappings!

  • Grant et al., 2011 (Bioinformatics)

    1. GSNAP performs as one of the best for mapping RNA-Seq data

  • 1. GSNAP results in more mapped tags compared to ELAND

    2. Splice sites (only known)

  • X CastM.m. castaneus

    129Inbred strainMix of M.m. DomesticusAnd M.m. musculus

    XCI in polymorphic mouse ES cells

    Use a predefined set of SNPs for assayingSNPs 129 vs Cast total (Sanger mouse genome project, 2010 + Perlegen study, 2007): 20.8 million(total genome 2,726 million bases; so each 1:131 bases)SNPs 129 vs Cast X chromosome: 0.6 million(X chromosome 166.6 million bases; so each 1:278 bases)

    X-chromosomes

    Xa (Cast)Xi (129)

    Cast129

    mRNA-Seq on polyA+ RNA

  • 3. SNP tolerant mapping is VERY important

    B6 (reference)129

    Cast2 million SNPs

    Various other studies map against both genomes and join these mappings later

    Genome-wide expectation:1:1 ratio between 129 and Cast(over all SNPs)

  • Features of mapped reads Allele specific reads

    Short summary GSNAP mappingMaps ~60% of QC reads uniquely to genome in mouse mRNA-Seq6-8% of these over slice sites8-12% allele specific

  • Massive allele specific mappings of the RNA-seq

    Stringent mappings•Only polymorphisms sequenced two times or more for both allelels•Minimal coverage of 80 tags on both alleles over the transcript over the timecourse•Standard error of the mean < 0.5 over the SNPs over a transcript over the summedtimecourse (slide)

  • Consistency allele specific mappings over gene

    A/T G/A C/G A/T304/385 350/418 201/248 562/510

    Standard error of the mean (SEM): 0.02; As < 0.1: gene included

    A/T G/A C/G409/433 944/877 398/1

    Standard error of the mean (SEM) : 0.13; As > 0.1: gene excluded

  • Allele specific RNA-seq show high accuracy for discriminating between both alleles

    129 allele Cast allele

    Chr17 (similar for other autosomes)

    Both alleles

    25% 100% 175%

    Allele specific informationTotal : 10455 of 14645 expressedgenes (71%)

    Genes on X: 391 of 559 (70%)

    Multiply gene ratio over complete transcript with RPKM: Expression profile over complete transcript

  • Thanks:Stefan Barakat (Erasmus MC, Rotterdam, NL)Joost Gribnau (Erasmus MC, Rotterdam, NL)Erik Splinter (Hubrecht Institute, Utrecht, NL)Wouter de Laat (Hubrecht Institute, Utrecht, NL)Jennifer Chow (Curie Institute, Paris, FR) Edith Heard (Curie Institute, Paris, FR)Alice Jouneau (INRA, Paris, FR)

    NCMLS Nijmegen, NL:Hinri KerstensKees-Jan FrançoisArjen BrinkmanHenk Stunnenberg

    Xa Xi

  • Main Figures

  • Main Figures


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