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The Genome Analysis Centre
Building Excellence in Genomics and Computational Bioscience
TILLING @ TGAC
Sarah Ayling
Crop Genomics and Diversity
[email protected]
The Genome Analysis Centre
Building Excellence in Genomics
and Computational Bioscience
The Genome Analysis Centre
The Genome
Analysis Centre
Definitions
Forward genetics is the approach of
determining the genetic basis responsible for a
phenotype – classical genetics
Reverse genetics is an approach to discover
the function of a gene by analyzing the
phenotypic effects of specific engineered gene
sequences.
TILLING: Targeting Induced Local Lesions in
Genomes
Source: Wikipedia
The Genome Analysis Centre
The Genome
Analysis Centre
TILLING by sequencing
Induce
genetic
variation
Base changes:
C > T; G > A GCGATGAGCTCGAAGACGCA
GCGATAAGCTCGAAGATGCA
Detect base
changes in
genes
Phenotype
plants with
changes in
your gene of
interest
Sequence once, database for everyone…
The Genome Analysis Centre
TILLING projects:
Brassica rapa
Triticum aestivum
The Genome Analysis Centre
The Genome
Analysis Centre
Triangle of U
black mustard
turnip, Chinese cabbage
broccoli, cabbage
oil seed rape, swede
Wikipedia (U, Jpn J Bot
7:389452, 1935)
The Genome Analysis Centre
The Genome
Analysis Centre
Triangle of U
black mustard
turnip, Chinese cabbage
broccoli, cabbage
oil seed rape, swede
Wikipedia (U, Jpn J Bot
7:389452, 1935)
The Genome Analysis Centre
The Genome
Analysis Centre
Mutant
Areas ofpopulation
Activity - brassica
Genotype: R-o-18
• Inbred line of B. rapa subsp. Trilocularis (Yellow Sarson)
• Closely related to B. rapa oilseed crops grown in Pakistan
EMS population developed in Lars Østergaard group, >1000 lines
Treat seed with EMS
(ethyl methanesulfonate)
(0.3 and 0.4%)
Grow M1 plants
(G:C to A:T
mutations)
Self-fertilise
and harvest
seed
Grow M2
plants
Harvest M3
seed
Extract DNA
Stephenson et al., BMC Plant Biology 2010
The Genome Analysis Centre
The Genome
Analysis Centre
Slide: Jon Wright
Polyploid wheat
The Genome Analysis Centre
The Genome
Analysis Centre
Polyploid wheat
The Genome Analysis Centre
The Genome
Analysis Centre
Mutant
Areas ofpopulation
Activity - wheat
Genotype:
Triticum aestivum cv Cadenza (Bread wheat)
EMS population developed in Andy Philips group,
>1500 lines as part of WGIN1
Treat seed with EMS
(ethyl methanesulfonate)
Grow M1 plants
(G:C to A:T
mutations)
Self-fertilise
and harvest
seed
Grow M2
plants
Harvest M3
seed
The Genome Analysis Centre
The Genome
Analysis Centre
Extract DNA
Exome capture
Bamshad et al., Nature Reviews Genetics, 2011
The Genome Analysis Centre
The Genome
Analysis Centre
B. rapa genome & annotation
The Genome Analysis Centre
B. rapa capture design
B. rapa genome
http://brassicadb.org, v1.2
41,019 protein-coding genes, 48 Mb total sequence
48 Mb
30 Mb
Conserved domain
CODDLE-like: Comai lab
(Codons Optimized to Detect Deleterious Lesions)
The Genome Analysis Centre
The Genome
Analysis Centre
Slide: Jon Wright
Sequencing strategy
•
•
•
•
Paired-end Illumina
Fragment size: 300bp
Read lengths: 2x150bp
24-plex run over 2 lanes
• = 12-plex
The Genome Analysis Centre
The Genome
Analysis Centre
Sequencing results
998
Number of samples processed
25,892,695
Mean number of reads per sample
3,755,166
Minimum number of reads for a sample
38.5
Mean target coverage
Mean percentage of targets with <2x coverage
Mean percentage of target bases with >=10x coverage
5.1
86.3
Target coverage variability between samples
120
60
Samples
0
The Genome Analysis Centre
The Genome
Analysis Centre
Slide: Jon Wright
MAPS pipeline
BWA mapping
Picard
MarkDuplicates
MAPS part1:
(whole batch)
minLib=n-2
minCov=10
maxCov=1000
hetOneMinPer=20
Mpileup
MAPS part2:
MAPS part1
MAPS part2
(per sample)
minLib=n-2
minCov=10
hetMinPer=30
homMinCov=3
hetMinCov=6
REF
x
x
x
x
x
x
x
x
line1
x
line2
line3
line4
line5
line6
line7
line8
Henry et al. 2014. Plant Cell
The Genome Analysis Centre
The Genome
Analysis Centre
Results
Mean number of mutations per sample
Mean mutation density
1188.7
1 per 49.9 kb
% Homozygous
10
% Heterozygous
90
Mean % EMS mutations (C:G->A:T)
99.1
ACGT
The Genome Analysis Centre
The Genome
Analysis Centre
Slide: Jon Wright
Results
92.2% of B. rapa genes contain
non-synonymous mutations
The Genome Analysis Centre
The Genome
Analysis Centre
Slide: Jon Wright
Accessing the data
Slide: Jon Wright
Accessing the data
Slide: Jon Wright
Accessing the data
Slide: Jon Wright
Coming soon to revgen.jic.ac.uk
The Genome Analysis Centre
Next steps?
• Order your seeds (multiple mutants per gene)
• Grow your plants
• Check phenotypes
• Cross/self to obtain homozygous alleles
• Check with KASP markers
• Grow your plants
• Check phenotypes
• Publish 
The Genome Analysis Centre
Acknowledgements
Brassica:
Jon Wright
Wheat:
Paul Bailey
Ksenia Krasileva
Ricardo RamirezGonzales
Platforms & Pipelines:
Leah Clissold
and team
Trevor Wang
Fran Robson
Lars Østergaard
Saleha Bakht
Andy
Phillips
The Genome Analysis Centre
Cristobal Uauy
Jorge Dubcovsky
Hans VasquezGross
Luca Comai
Meric Lieberman
Isabelle Henry