NimbleGen targeted resequencing

1. Use 20 μg of genomic DNA
2. Fragmentize DNA to optimal fragment length of 350 bp (range approx. between 100-800 bp) using sonication.
3. Use QIAguick PCR purification Kit for purifying the sample. Split each sample between 4 columns while purifying and elute each one of them in 30 μl volume and pool (i.e. final volume 4x30=120 μl)
4. Blunt-end fragments using:
   DNA sample 120 μl
   Water (nuclease free) 208.5 μl
   T4DNA ligation buffer (NEB) 40 μl
   dNTPs (10mM each) 16 μl
   T4DNA Polymerase (Fermentas) (5u/μl) 10 μl
   Klenow Fragment (Fermentas) (5u/μl) 0.5 μl
   T4 PNK (polynucleotide kinase) (Fermentas) (10u/μl) 5 μl Incubate 30 min at 25°C following 30min at 37°C
   Purify the sample as described in step 3, elute each column in 32 μl (final volume 4x32=128 μl)
5. Add A-overhangs using:
   DNA sample 128 μl
   Water (nuclease free) 4 μl
   Klenow buffer (NEB2) 20 μl
   dATP (1mM) 40 μl
   Klenow exo- (Fermentas) (5u/μl) 8 μl
   Incubate 40 min at 37°C
   Use MinElute PCR purification Kit for purifying the sample. Split each sample between 4 columns while purifying and elute each one of them in 10 μl volume and pool (i.e. final volume 4x10=40 μl)
6. Ligate adapters using:
   DNA sample 40 μl
   Water (nuclease free) 25 μl
   DNA Ligase buffer (Fermentas) 10 μl
   Adapter Oligo Mix (Illumina) 15 μl
   T4 DNA Ligase (Fermentas) (5u/μl) 10 μl
   Incubate 2 hours at 25°C
   Purify the sample as described in step 3, elute each column in 30 μl (final volume 4x30=120 μl)

Hybridization, washing and elution of captured DNA library is performed following manufacturers' instructions.

1. Uncaptured DNA library amplification (uncaptured LM-PCR)
   a. Use 1 μl of fragment library and dilute it with 103 μl of nuclease free water
   b. Start the amplification using:
   Phusion High-Fidelity PCR MasterMix 50 μl
   ILL PCR1 primer (40μM) 2 μl
   ILL PCR2 primer (40μM) 2 μl
   DNA 46 μl
   PCR-program: 98°C for 30 sec following 30 cycles of 98°C for 10 sec, 65°C for 30 sec and 72°C for 30 sec followed by final elongation step of 72°C for 5 min.
   PCR product can be checked on agarose gel, to see a similar smear as after genomic DNA sonication step.
   PCR products should be purified using QIAquick PCR Purification Kit. Split PCR product between 2 columns while purifying and elute each one of them in 30 μl volume and pool (i.e. final volume 2x30=60 μl). Measure concentration and dilute purified product to 5 ng/μl.
2. Captured DNA library amplification (captured LM-PCR)
   a. Speedvac the eluted captured fragment library until completely dry
   b. Redissolve DNA in 320 μl nuclease free water. Vortex and spin. Incubate at 70°C for 10 min. Vortex and spin.
   c. Optimize the LM-PCR cycle number by setting up a following PCR for each sample:
   Phusion High-Fidelity PCR MasterMix 20 μl
   ILL PCR1 primer (40μM) 0.8 μl
   ILL PCR2 primer (40μM) 0.8 μl
   DNA 18.4 μl
   PCR-program: use the same program as described for uncaptured LM-PCR with a total cycle number of 26. Pause the PCR-machine and remove 5 μl of PCR-product from each sample after 14, 18, 20, 22, 24 and 26 cycles. Run PCR-products on agarose gel in order to determine the cycle number necessary for amplifying the fragment library for sequencing. One should pick the cycle number when the smear of amplified library has just become visible on the gel. This is necessary for avoiding overamplification of most abundant fragments.
   d. Run the captured DNA LM-PCR in 8x50 μl PCRs using:
   Phusion High-Fidelity PCR MasterMix 25 μl
   ILL PCR1 primer (40μM) 1 μl
   ILL PCR2 primer (40μM) 1 μl
   DNA 23 μl
   PCR-program: use the same program as described for uncaptured LM-PCR with a cycle number determined at the optimization step. Pool PCR-products for the same sample and purify using QIAguick PCR Purification Kit. Split the sample between 3 columns and elute each in 30 μl (i.e. final volume 3x30=90 μl). Measure concentration and dilute an aliquot of purified product to 5 ng/μl.

PCR should be run for both uncaptured and captured DNA of each sample and preferably in triplicates. PCR-primers should be designed for targeted (for evaluating the enrichment) and not-targeted (for evaluating the specificity of hybridization) regions (preferably at least 2 primer-pairs for each).

1. Set up a Real-Time PCR using:
   SYBR Green PCR MasterMix 7.5 μl
   primerF (10μM) 0.5 μl
   primerR (10μM) 0.5 μl
   Water 5.5 μl
   DNA (5ng/μl) 1 μl
   PCR-program: use one that is suitable for your primers.
2. Evaluate the enrichment factor using a deltaCt-method i.e. evaluate the cycle number difference in the exponential phase of the PCR between uncaptured and captured sample. If the enrichment factor is above 200 (deltaCt>7 or 8) the sample is considered as successfully enriched and ready for next-generation sequencing.
3. Evaluate the hybridization specificity by looking at the amplification with the not-targeted primer-pairs. In this case the captured DNA samples should show no or very weak amplification.