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Transcript 
RAD – technology overview
Baird et al. 2008 PLoS ONE
P1-adaptor & multiplexing
Sequencing
primer site
Restriction
site
Sample 1
Forward
amplification
primer site
Barcode
Sample 2
Sample 3
Ligate P1
adaptor to
digested
gDNA
Pool barcoded
samples and
shear
Size
selection
P2-adaptor & PCR selection
P2 ligation
Sequencing
primer site
Complement of
reverse amplification
primer site
PCR
selection
Size
selection
Trouble-shooting & optimisation
•
•
•
•
•
•
Quality of DNA
Quality of adaptors
P1:DNA ratio
Choice of ligase
Sonication
Quality control
Trouble-shooting & optimisation
• Quality of DNA
– Use RNase in extraction
– Quantification – Nanodrop vs fluorometer
– Check integrity on a gel
– Clean up with column if necessary
Trouble-shooting & optimisation
• Quality of DNA
• Quality of adaptors
– phosphorothioate bond
– purification
• P1:DNA ratio
– too little P1 – poor
library amplification
– too much P1 –
concatemers take over
£££££
Collaborate!
Trouble-shooting & optimisation
•
•
•
•
Quality of DNA
Quality of adaptors
P1:DNA ratio
Choice of ligase
– not everything works in NEB Buffer 2
• Sonication
– tight band in desired size range
• Quality control
– clone and Sanger sequencing
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