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The document discusses testing whether whole genome bisulfite sequencing (WGBS) can be used to discover methylated cytosines in non-model species with limited genomic resources. It describes conducting a feasibility trial using Atlantic salmon sequences as a surrogate for the oyster genome. Short reads were generated from the salmon sequences, converted to simulate bisulfite treatment, and assembled using various tools. The results showed it was difficult to map methylated regions due to the limited reference genome available. While WGBS may be possible, improved assembly tools and reduced representation approaches are needed.

Technology

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.

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

$Next Steps   Find tool to do ‘customizable’ assembly   e.g. only allow C/T (or G/A mismatches)   new protocol using SOLiD that will only sequence 1 strand (this will make analysis easier)   reduced representation   digest w/ restriction enzymes and size select DNA prior to making library   DNA methylation enrichment kit – fractionate DNA by binding to methyl binding domain proteins (only sequence heavily methylated regions)$

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