Mouse Genomes Project + RNA-Editing

Mouse genomic variation and its effect on
phenotypes and gene regulation

Thomas Keane
Vertebrate Resequencing Informatics

Wellcome Trust Sanger Institute,
Hinxton,
Cambridge,
UK

NUI Maynooth 20th April, 2012

Mouse genomic variation and its effect on phenotypes
and gene regulation

 Mouse Genomes Project

 RNA-Editing


Sequencing Technologies over past 30 years

MR Stratton et al. Nature 458, 719-724 (2009)


Sanger total sequence (2007-2009)
Gbp


Sanger total sequence to-date

HiSeq 2000
Gbp


The Laboratory Mouse


Mouse Genome Project (2002)


International Knockout Mouse Consortium


Large Outbred Crosses

Founder set of inbred strains and randomly cross

  Heterogeneous stock
  Collaborative Cross

Large numbers of resulting mice
  Comprehensively phenotyped
  Recurrent phenotypes assessed
  Identify QTL regions
 Knowing the origin of haplotype blocks
Collaborative Cross Consortium (2009) Genetics

Full sequence variation of founder mice required to find potential
causitive mutations


Mouse Genomes Project

Sequencing 18 laboratory mouse strains
  Largest effort to date to sequence genomes of laboratory mouse strains

Primary goals
  Deep sequencing of each strain (>25x)
  Comprehensive catalog sequence variation

What sort of variation?
  SNPs – single base changes (A->G etc.)
  Indels – insertions or deletions of a few bases
  Structural variation – larger structural differences

Illumina sequencing platform
  Raw data was generated in 2009
  Approx. ~1.2Tbp


What does the data look like?

Whole-genome shotgun (WGS)

Sequence in parallel ends of
millions of fragments
  300-500bp in size
  Read sequence of 100bp of
either end

Reference


Variation Catalog

Keane et al (2011) Nature


Variation Catalog


Results of the project

Genomic variation and its effect on phenotypes

Jonathan Flint & Richard Mott
Keane et al (2011) Nature




Structural variation catalog

Binnaz Yalcin, Kim Wong, Thomas Keane, Jonathan Flint
Yalcin et al. (2010) Nature




SVMerge

Structural variation methods

Wong, Keane, Stalker, Adams (2010) Gen Biol






Novel structural variation types

Binnaz Yalcin & Kim Wong
Yalcin et al. (2012) Gen Biol






Transposable elements

Nellaker, Keane, Wong et al., under review







Transposable elements

RNA-Editing…….


RNA-Editing

Site-selective post-transcriptional alteration of double-stranded RNA

Adenosine deaminase acting on RNA (ADAR) family of enzymes
  Adenosine residues to inosines
  Observe A-to-G SNPs in cDNA

ADARs
  Bind to double-stranded regions of RNA
  Modify multiple neighbouring adenosines

Apobec-1 mediated C-to-U RNA editing

Novel source of protein isoform diversity
Wulff and Nishikura (2009) WIREs RNA
  HTR2C gene: five edit sites lead to 28 mRNAs


HTR2C gene

Wahlstedt et al (2009) Gen Res


RNA-Seq

Isolate RNA and reverse transcribe to cDNA
  Fragment cDNA and directly sequence
  No reference bias and huge dynamic range

Uses
  Gene expression analysis
  Transcript discovery and annotation new genomes
  Alternative splicing

RNA-editing McIntyre et al (2011) BMC Gen

  Align the RNA-seq reads to the reference genome
  If the bases disagree with the genomic sequence data at the
corresponding position…..


RNA-Editing?

RNA-seq
Replicate 1

RNA-seq
Replicate 2

DNA


Human RNA-Editing

Li et al. (2011) Science


RNA-Seq is not the same as genomic sequencing

Alignment of RNA-Seq reads is not trivial
  Most genomic short read aligners are not splice aware

RNA-seq
Replicate 1

RNA-seq
Replicate 2

DNA


RNA-Seq is not the same as genomic sequencing

What about processed pseudo-genes?

Pink et al (2011) RNA

cDNA fragment Pseudogene

Functioning gene Exon 1 Exon 2

Exon 1 Exon 2


What about in mouse?

  RNA-Seq of 15 mouse strains
  Whole-brain tissue
  2-4 biological replicates per strain
  ~5Gbp per replicate

Previous catalogs
  Neeman et al.
  Zaranek et al. - several tens of gigabases of human and mouse cDNA
sequence
  Rosenberg et al. - RNA-seq for C57BL/6J strain

Hindered by lack of corresponding genomic sequencing

We generated
  Deep whole genome sequencing
  Corresponding RNA-Seq from whole-brain tissue across 15 strains
 2-4 biological replicates


Our Pipeline

gDNA cDNA Splice-aware
SNVs SNVs
realignment
Minimum Depth 10x
31,923 sites

304,817 candidate sites 98,061 unambiguous sites Filtering Replicate Consistency
62,889 sites

Estimated FDR 2.9%
No assumptions about the
nature of editing made
Assumed editing by ADARs 5,579 filtered sites
which usually occurs in clusters End Distance Bias
59,775 sites

One-type mismatch
Cluster extension Strand Bias
clusters added
42,238 sites

Variant Distance Bias
7,389 final sites 7,133 sites 36,213 sites


Effect of Filtering Strategy


Validation

Sequenom validation
  Random set of 611 calls from both the filtered set of 5,579 RNA editing
sites
  19 non A-to-G editing sites raw calls -> all confirmed false positives
  Discrepancy rate of 10.5%
 Enriched at positions where editing level is <20%

T-to-C editing
  Novel form of RNA-editing?
 Uncertainties in strand assignment of transcripts
 Result of calls made in antisense transcripts, mis-annotations

Assuming all non A-to-G edits are false
  False-discovery rate of our call set is 2.9%


Striking Conservation


Editing Levels


Genomic Context


Protein Coding Edits

23 previously known non-synonymous coding edits

Extended this by a further 30 sites
  24 were by Sanger sequencing of cDNA

Cacna1d gene
  Encodes the Cav1.2 voltage-
gated calcium channel
  Known to undergo extensive
alternative splicing
  Two novel non-synonymous
edits
  Capillary sequencing validation
  Observed 3 different transcripts


Cacna1d


Rare C-to-U Edit: Mfn1


Cds2 - UTR
D R
a g
a g
a g
a g
a g
a g
a g
a g
a g
a g
a g
a g
a g
a g
a g
g g
g g
g g

Rat g g
RNA-editing appears to revert genomic sequence back to ancestral state

Mice homozygous for disruptions in this gene display a lethal phenotype

Several known across-species examples
  RNA-editing maintaining conservation at the protein level despite genomic sequence divergence


Human Follow-up Studies

Li et al (2011) Science

Ramaswami et al. (2012) Nat Meth

Bahn et al (2011) Gen Res
Peng et al (2011) Nat Bio


To do

First phase of the project was cataloging variation

Full denovo assemblies of the strains
  Generating higher quality sequencing data for the 18 strains
  Long fragment end sequencing – 3, 6, 10, 40kb fragments

De novo assembly
  Discover novel haplotypes
  Novel gene structures in the divergent strains

Mouse pan-genome
  Reference bias
  New mouse reference genome graph
 Including novel non-reference haplotypes shared amongst subsets of the
strains


Acknowledgements and Questions

  Sanger Insitute
  David Adams, Petr Danecek, Kim Wong, Guy Slater, Sendu Bala et al.
  Wellcome Trust Center for Human Genetics
  Jonathan Flint, Binnaz Yalcin, Richard Mott, Leo Goodstadt et al.
  EBI
  Ewan Birney
David Adams
  University of Oxford
  Chris Ponting, Chris Nellaker, Andres Heger, Grant Belgard
RNA-Editing
  Petr Danecek, David Adams, Chris Nellaker

Jonathan Flint

Email: thomas.keane@sanger.ac.uk


WTSI PhD Programme


Mouse Genomes Project + RNA-Editing

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