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Whole Genome
Bisulfite Sequencing
     (feasibility trial)
          FISH 546
       Mackenzie Gavery
Introduction
   QUESTION:
   is whole genome bisulfite sequencing (WGBS) a viable option
   for discovering methylated cy...
Introduction
   QUESTION:
   is whole genome bisulfite sequencing (WGBS) a viable option
   for discovering methylated cy...
Background: bisulfite sequencing

                                    m
        C AT G T TA C G AT C G G C T C G
         ...
Bisulfite-PCR
  previous work – use design primers to amplify
  specific regions of interest




                        ...
WGBS Challenges:
    sequencing issues – sequencers can have problems
    w/ low complexity sequence

    non-model spec...
Approach:
  generate mock bisulfite-seq reads using Atlantic
  salmon GSS sequences as surrogate to C.gigas

  use CLC t...
Approach:

 Atlantic salmon         after de novo        generate 1 million
  GSS: 203,387        assembly: 128,337       ...
Assembly 1st try:

                                          assemble          BLAST non
                           de nov...
Analysis summary:

                           non-treated         non-treated        non-treated
                         ...
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 cytos...
Conclusions:
  QUESTION:
  is whole genome bisulfite sequencing (WGBS) a viable
  option for discovering methylated cytos...
Next Steps
   Find tool to do ‘customizable’ assembly
     e.g. only allow C/T (or G/A mismatches)

   new protocol usi...
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Gavery Fish546

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Transcript of "Gavery Fish546"

  1. 1. Whole Genome Bisulfite Sequencing (feasibility trial) FISH 546 Mackenzie Gavery
  2. 2. 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.
  3. 3. 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.
  4. 4. 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
  5. 5. Bisulfite-PCR   previous work – use design primers to amplify specific regions of interest Kismeth   challenging to design primers with specificity, limited to known sequences
  6. 6. 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
  7. 7. 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
  8. 8. 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)
  9. 9. 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
  10. 10. 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
  11. 11. Other tools: Nature Reviews Genetics 11, 191-203 | doi:10.1038/nrg2732
  12. 12. 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
  13. 13. 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
  14. 14. 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)
  15. 15. Thank you
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