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

Reduced representation libraries

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1. Genotyping by Sequencing (GBS)2. Hybridization capture techniques

(hyRAD)3. epi-GBS (methylation GBS)

Terminology and methodology

3Andrews et al (2016) Nat Rev Genet

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

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

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

8Andrews et al (2016) Nat Rev Genet

GBS vs RAD & ddRAD

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

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’

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’

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?

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