RAD – technology overview Baird et al PLoS ONE.

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Presentation transcript:

RAD – technology overview Baird et al PLoS ONE

P1-adaptor & multiplexing Ligate P1 adaptor to digested gDNA Pool barcoded samples and shear Size selection Forward amplification primer site Sequencing primer site Barcode Restriction site Sample 1 Sample 2 Sample 3

P2-adaptor & PCR selection P2 ligation PCR selection Size selection Complement of reverse amplification primer site Sequencing primer site

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