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FISH 546 Mar 2010:  Whole Genome Bisulfite Sequencing (feasibility trial)
 

FISH 546 Mar 2010: Whole Genome Bisulfite Sequencing (feasibility trial)

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FISH 546: Aquatic-BioInformatics

FISH 546: Aquatic-BioInformatics

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    FISH 546 Mar 2010:  Whole Genome Bisulfite Sequencing (feasibility trial) FISH 546 Mar 2010: Whole Genome Bisulfite Sequencing (feasibility trial) Presentation Transcript

    • Whole Genome Bisulfite Sequencing (feasibility trial) FISH 546 Mackenzie Gavery
    • Introduction   QUESTION: is whole genome bisulfite sequencing (WGBS) a viable option for discovering methylated cytosines in non-model species with limited genomic resources?   HYPOTHESIS: With limited reference sequence available, it will be very difficult to annotate methylated regions of DNA   WHO CARES: DNA methylation is an epigenetic mechanism with important regulatory functions. Evidence for regulatory role in oysters, would like to explore in diff populations / generations but need to know where to look.
    • Introduction   QUESTION: is whole genome bisulfite sequencing (WGBS) a viable option for discovering methylated cytosines in non-model species with limited genomic resources?   HYPOTHESIS: With limited reference sequence available, it will be very difficult to annotate methylated regions of DNA   WHO CARES: DNA methylation is an epigenetic mechanism with important regulatory functions. Evidence for regulatory role in oysters, would like to explore in diff populations / generations but need to know where to look.
    • Background: bisulfite sequencing m C AT G T TA C G AT C G G C T C G bisulfite m U AT G T TA U G AT C G G U T C G PCR T AT G T TA T G AT C G G T T C G ATA C A AT A C TA G C C AT G C
    • Bisulfite-PCR   previous work – use design primers to amplify specific regions of interest Kismeth   challenging to design primers with specificity, limited to known sequences
    • WGBS Challenges:   sequencing issues – sequencers can have problems w/ low complexity sequence   non-model species genomic resources limited   C.gigas   Most resources are ESTs (coding sequences only)   bioinformatics   assemblies/alignments need to recognize C/T conversion   bisulfite treatment results in 4 unique strands after PCR
    • Approach:   generate mock bisulfite-seq reads using Atlantic salmon GSS sequences as surrogate to C.gigas   use CLC to assemble mock bisulfite treated reads back to non-treated mock sequences
    • Approach: Atlantic salmon after de novo generate 1 million GSS: 203,387 assembly: 128,337 random, ~40bp sequences contigs fragments create similar convert all C to T, use the non-treated fragment library that with exception of library to assemble is not converted to ‘ACG’ sequences bisulfite treated use as reference (259,750 ‘C’s’ reads sequence remain)
    • Assembly 1st try: assemble BLAST non de novo non treated fragments bisulfite reads treated contigs assembly non to de novo non with matches treated treated for ID 1 million 459 contigs 42 contigs Found hits, short reads (~300bp) (~ 46bp) but many 40 mil bp not 1940 bp annotated
    • Analysis summary: non-treated non-treated non-treated reference A* reference B reference converted assembly settings: limit=8 limit=8 limit=8 (‘global alignment’, mismatch cost =2 mismatch cost =3 mismatch cost =3 ‘allow mismatch) score limit = 8 score limit = 15 score limit = 15 contigs generated 42 71 11,213 (total bp) (1940 bp) (21,487) (508,799) total SNPs 42 42 473
    • Other tools: Nature Reviews Genetics 11, 191-203 | doi:10.1038/nrg2732
    • Conclusions:   QUESTION: is whole genome bisulfite sequencing (WGBS) a viable option for discovering methylated cytosines in non- model species with limited genomic resources?   HYPOTHESIS: With limited reference sequence available, it will be very difficult to map methylated regions of DNA   ANSWER: Yup
    • Conclusions:   QUESTION: is whole genome bisulfite sequencing (WGBS) a viable option for discovering methylated cytosines in non- model species with limited genomic resources?   HYPOTHESIS: With limited reference sequence available, it will be very difficult to map methylated regions of DNA   ANSWER: Yup
    • Next Steps   Find tool to do ‘customizable’ assembly   e.g. only allow C/T (or G/A mismatches)   new protocol using SOLiD that will only sequence 1 strand (this will make analysis easier)   reduced representation   digest w/ restriction enzymes and size select DNA prior to making library   DNA methylation enrichment kit – fractionate DNA by binding to methyl binding domain proteins (only sequence heavily methylated regions)
    • Thank you