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Using long-read sequencing to detect imprinted DNA methylation

View ORCID ProfileScott Gigante, View ORCID ProfileQuentin Gouil, View ORCID ProfileAlexis Lucattini, View ORCID ProfileAndrew Keniry, Tamara Beck, Matthew Tinning, View ORCID ProfileLavinia Gordon, View ORCID ProfileChris Woodruff, View ORCID ProfileTerence P. Speed, View ORCID ProfileMarnie E. Blewitt, View ORCID ProfileMatthew E. Ritchie
doi: https://doi.org/10.1101/445924
Scott Gigante
aThe Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052 Australia
eDepartment of Genetics, Yale University, New Haven, 06520 USA
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Quentin Gouil
aThe Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052 Australia
bDepartment of Medical Biology, The University of Melbourne, Parkville, 3010 Australia
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Alexis Lucattini
cAustralian Genome Research Facility, 305 Grattan Street, Melbourne, 3000 Australia
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Andrew Keniry
aThe Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052 Australia
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Tamara Beck
aThe Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052 Australia
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Matthew Tinning
cAustralian Genome Research Facility, 305 Grattan Street, Melbourne, 3000 Australia
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Lavinia Gordon
cAustralian Genome Research Facility, 305 Grattan Street, Melbourne, 3000 Australia
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Chris Woodruff
aThe Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052 Australia
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Terence P. Speed
aThe Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052 Australia
dSchool of Mathematics and Statistics, The University of Melbourne, Parkville, 3010 Australia
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Marnie E. Blewitt
aThe Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052 Australia
bDepartment of Medical Biology, The University of Melbourne, Parkville, 3010 Australia
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Matthew E. Ritchie
aThe Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052 Australia
bDepartment of Medical Biology, The University of Melbourne, Parkville, 3010 Australia
dSchool of Mathematics and Statistics, The University of Melbourne, Parkville, 3010 Australia
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Abstract

Systematic variation in the methylation of cytosines at CpG sites plays a critical role in early development of humans and other mammals. Of particular interest are regions of differential methylation between parental alleles, as these often dictate monoallelic gene expression, resulting in parent of origin specific control of the embryonic transcriptome and subsequent development, in a phenomenon known as genomic imprinting.

Using long-read nanopore sequencing we show that, with an average genomic coverage of approximately ten, it is possible to determine both the level of methylation of CpG sites and the haplotype from which each read arises. The long-read property is exploited to characterise, using novel methods, both methylation and haplotype for reads that have reduced basecalling precision compared to Sanger sequencing. We validate the analysis both through comparison of nanopore-derived methylation patterns with those from Reduced Representation Bisulfite Sequencing data and through comparison with previously reported data.

Our analysis successfully identifies known imprinting control regions as well as some novel differentially methylated regions which, due to their proximity to hitherto unknown monoallelically expressed genes, may represent new imprinting control regions.

  • List of abbreviations

    AUROC
    Area Under Receiver Operating Characteristic curve
    CpG
    5’-C-phosphate-G-3’
    CGI
    CpG island
    DMR
    differentially methylated region
    DML
    differentially methylated locus
    F1
    first filial generation
    FDR
    false discovery rate
    ICR
    imprinting control region
    HMM
    Hidden Markov Model
    ONT
    Oxford Nanopore Technologies
    PB
    Pacific Biosciences
    RRBS
    reduced representation bisulfite sequencing
    SNP
    single nucleotide polymorphism
    TSS
    transcriptional start site
  • Copyright 
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    Posted October 17, 2018.
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    Using long-read sequencing to detect imprinted DNA methylation
    Scott Gigante, Quentin Gouil, Alexis Lucattini, Andrew Keniry, Tamara Beck, Matthew Tinning, Lavinia Gordon, Chris Woodruff, Terence P. Speed, Marnie E. Blewitt, Matthew E. Ritchie
    bioRxiv 445924; doi: https://doi.org/10.1101/445924
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    Using long-read sequencing to detect imprinted DNA methylation
    Scott Gigante, Quentin Gouil, Alexis Lucattini, Andrew Keniry, Tamara Beck, Matthew Tinning, Lavinia Gordon, Chris Woodruff, Terence P. Speed, Marnie E. Blewitt, Matthew E. Ritchie
    bioRxiv 445924; doi: https://doi.org/10.1101/445924

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