Genomic DNA Sample Preparation 1.Nebulisation of genomic DNA Double stranded genomic DNA is broken into random sized fragments of between 100 to 1000bp 2. Perform End Repair 5’-AGTCTTGGATCGACTCT-3’ 3’-ACCTAGCTG-5’ Convert the overhangs from fragmentation into Blunt ends, using T4 polymerase & E. coli DNA Polymerase I Klenow fragment The 3’ to 5’ exonuclease activity of these enzymes removes 3’ overhangs & the polymerase activity fills in the 5’ overhangs P5’-AGTCTTGGATCGAC-3’ 3’-TCAGAACCTAGCTG-5’P

Genomic DNA Sample Preparation 3. Add ‘A’ Bases to the 3’ End of the DNA Fragments P5’-AGTCTTGGATCGAC-3’ 3’-TCAGAACCTAGCTG-5’P An ‘A’ base is added to the 3’ end of the blunt phosphorylated DNA fragments, using the polymerase activity of Klenow fragment (3’ to 5’ exo minus). This prepares the DNA fragments for ligation to the adapters, which have a single ‘T’ base overhang at their 3’ end P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P 4. Ligate Adapters to the DNA Fragments P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P Adapters are ligated to the ends of the DNA fragments, preparing them to be Hybridized to the flow cell P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P

Genomic DNA Sample Preparation 5. Purify Ligation Products The products from the ligation are purified on a 2% agarose gel, to remove all unligated adapters, Remove any adapters that may have ligated to one Another, & select a size range of templates to go on The cluster generation platform You can select more than one size-range of Adapter-ligated DNA by excising slices from different parts of the gel. A short insert template is 150-200bp, while 300-650bp is a long insert template

Genomic DNA Sample Preparation 6. Enrich the Adapter-Modified DNA Fragments by PCR PCR is used to selectively enrich those DNA fragments that have adapter molecules on both ends, & to amplify the amount of DNA in the library. The PCR is performed with two primers that anneal to the ends of the adapters P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P Denaturation P5’-AGTCTTGGATCGACA-3’ Primer Annealing Amplify using the following PCR protocol: *30 seconds at 980C *18 cycles of: 10 seconds at 980C 30 seconds at 650C 30 seconds at 720C *5 minutes at 720C *Hold at 40C 3’-ATCAGAACCTAGCTG-5’P Extension P5’-AGTCTTGGATCGACA-3’ 3’-NNNNNTCAGAACCTAGCTGTNN-5’ 5’-NNTAGTCTTGGATCGACNNNNN-3’ 3’-ATCAGAACCTAGCTG-5’P

Genomic DNA Sample Preparation 1 2 3 7. Validate the Library Perform the following quality control steps on the DNA Library: Determine the library concentration by measuring its absorbence at 260nm. The yield from the protocol should be between 500-1000ng of DNA. Measure the 260/280 ratio. It should be ~1.8-2.0. Either load 10% of the volume of the library on a gel and check that the size range is as expected, or run the DNA library on an Agilent 2100 Bioanalyzer. Determine the molar concentration of the library ready for Cluster Generation 1: Low molecular weight ladder 2: Long insert DNA library 3: Short insert DNA library