Mouse Genomes Project + RNA-Editing

NUI Maynooth 20th April, 2012

Mouse genomic variation and its effect on phenotypes and gene regulation

Thomas Keane Vertebrate Resequencing Informatics

Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK



 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) G

bp


Sanger total sequence to-date G

bp

HiSeq 2000


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

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

Collaborative Cross Consortium (2009) Genetics


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





Structural variation methods

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

SVMerge






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

 Mouse Genomes Project


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  HTR2C gene: five edit sites lead to 28 mRNAs Wulff and Nishikura (2009) WIREs RNA


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  Align the RNA-seq reads to the reference genome   If the bases disagree with the genomic sequence data at the

corresponding position…..

McIntyre et al (2011) BMC Gen


RNA-Editing?

RNA-seq Replicate 1

RNA-seq Replicate 2

DNA


Human RNA-Editing

Li et al. (2011) Science


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?

cDNA fragment

Exon 1 Exon 2

Pseudogene

Exon 1 Exon 2

Functioning gene

Pink et al (2011) RNA


What about in mouse?

Mouse Genomes Project  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 SNVs

cDNA SNVs

304,817 candidate sites 98,061 unambiguous sites

Splice-aware realignment

Filtering

Minimum Depth 10x 31,923 sites

Replicate Consistency 62,889 sites

End Distance Bias 59,775 sites

Strand Bias 42,238 sites

Variant Distance Bias 36,213 sites

5,579 filtered sites

Cluster extension One-type mismatch clusters added

No assumptions about the nature of editing made

Assumed editing by ADARs which usually occurs in clusters

7,133 sites 7,389 final sites

Estimated FDR 2.9%


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


Rare C-to-U Edit: Mfn1


Cds2 - UTR

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

D a a a a a a a a a a a a a a a g g g

R g g g g g g g g g g g g g g g g g g

Rat g g


Human Follow-up Studies

Ramaswami et al. (2012) Nat Meth

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

Li et al (2011) Science


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

David Adams

Mouse Genomes Project  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

 University of Oxford  Chris Ponting, Chris Nellaker, Andres Heger, Grant Belgard

RNA-Editing   Petr Danecek, David Adams, Chris Nellaker

Jonathan Flint

Email: [email protected]


WTSI PhD Programme

Education

Mouse Genomes Project + RNA-Editing