Gavery FISH546 Presentation

1. Whole Genome
Bisulfite Sequencing
(feasibility trial)
FISH 546
Mackenzie Gavery

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. 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. 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. 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. 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. 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. 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. 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. 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. Other tools:
Nature Reviews Genetics 11, 191-203 | doi:10.1038/nrg2732

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. 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. 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. Thank you