genotyping-by-sequencing; gbs, capture-rad and...
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Genotyping-by-Sequencing; GBS, capture-RAD and epi-GBS
Niccy Aitken1 and George Olah2
1 EcoGenomics and BioInformatics Lab, RSB, ANU2 Fenner School of Environment and Society, ANU
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Reduced representation libraries
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1. Genotyping by Sequencing (GBS)2. Hybridization capture techniques
(hyRAD)3. epi-GBS (methylation GBS)
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Terminology and methodology
3Andrews et al (2016) Nat Rev Genet
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1. Genotyping-by-Sequencing (GBS)
• Rob Elshire (Elshire et al 2011; Buckler lab, Cornell)
• Simultaneous SNP discovery & genotyping• Uses restriction enzyme to reduce genome
complexity and avoid repetitive fractions.• Technically very simple, scales very well
(I routinely pool 384, sometimes more)
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Overview of GBS
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Loss of cut site
Adapter A
Adapter B
“sticky ends”Barcode(4-8bp)
Barcode(4-8bp)
Flowcell binding seq Sequencing
primer
unknown sequence
Common enzymes:PstI 5’CTGCAG3’
ApeKI 5’GmCWGC3’
MspI/ 5’CCGG3’
HpaII 5’CCmGG3’
Computational Biology Service Unit, Cornell University
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GBS work flow
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(can be simultaneous)
RE
(I’ve taken to pooling after PCR)
8. Sequence J
Elshire et al, 2011, PLOS One
7. Evaluate fragment sizes
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Cost of in-house GBS
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$5.50 $5.50 $4.98 $4.98 $4.98
$26.85
$16.18
$10.32$8.54 $7.65
$0.00
$5.00
$10.00
$15.00
$20.00
$25.00
$30.00
0 1 2 3 4 5 6 7 8 9
Cos
t per
sam
ple
Number of plates pooled
GBS per sample cost
Lib prep alone
Lib prep plus NextSeq 75PE
Typically:Start with >100,000s SNPsTrim to >10,000s SNPs
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8Andrews et al (2016) Nat Rev Genet
GBS vs RAD & ddRAD
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GBS and DNA quality
• The most significant GBS/RAD limitation:– Requires large amounts of high quality high
mol weight DNA (ie intact restriction sites)– Therefore, not well suited to degraded, trace,
or museum specimens
• Turn to hybridisation capture techniques; hyRAD
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2. Hybridisation capture (hyRAD) Suchan et al 2016
• Prepare shotgun library of all “template” samples• Generate RAD based probes from high quality
DNA representing entire study• Use the RAD probes to hybridise and capture
target sequences • Doesn’t rely on intact restriction sites• Large sets of homologous loci from museum
specimens, without any a priori genome info
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2. Hybridisation capture (hyRAD)
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• Does not lose precious bp from 5’ exonuclease end repair
• More suited to low DNA concentrations because maintains both strands
Suchan et al (2016) PLoS OneTin et al (2014) PLoS One
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3. epi-GBS
• Analysis of genome-wide methylation profiles
• Similar to “normal” GBS except allows for identification of mCpG
• Differences– bisulphite conversion of ligated DNA– uses methylated adapters– PCR with uracil tolerant enzyme & mix
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3. epi-GBS (methylation)
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Original: ^C T C GG T A CG G A G C GA T G
^C ^C^C
C C T U G CG T A UG G A G U GC A T G
WC
WCBisulphite:
G G A A C GC A T APCR:C C T T G CG T A TW
W’
G G A G T GC A T GCC C T C A CG T A CC’
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epi-GBS; imputing original sequence
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Watson Crick True NucleotideA A A on WatsonT T T on WatsonG A G on WatsonT C C on WatsonC C mC WatsonG G mC Crick
C C T T G CG T A T
C C T C A CG T A C
W
C’
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Genotyping-by-Sequencing; GBS, capture-RAD and epi-GBS• Cost effective• Technically straight forward• Easily scalable• Generate 100,000s of SNPs for pop gen analysis
Routine pop gen: GBSMuseum / degraded samples: hyRADEpigenetic analysis: epi-GBS
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http://biology.anu.edu.au/research/facilities/ ecogenomics-and-bioinformatics-lab-ebl
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Questions – GBS and/or EBL?
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2. Hybridisation capture (hyRAD)
17Suchan et al (2016) PLoS OneTin et al (2014) PLoS One
• Does not lose precious bpfrom 5’ exonuclease end repair
• More suited to low DNA concentrations because maintains both strands