OP-HiSeq2000-sequencing-cBot-v2.5

Step 1. Prepare Sample DNA for Cluster Generation

Denature the template DNA with 2 N NaOH to a final DNA concentration [1] and final NaOH concentration of 0.1 N [2]. Incubate for 5 minutes at room temperature. Dilute the denatured DNA to titration series of [DNA_concentrations] pM with pre-chilled hybridization buffer and dispense 120 µl each into an 0.2 ml eight-strip tube.

Step 2. Cluster Generation

Set the denatured template DNA in eight-strip tube, and add reagents required for cluster generation and flow cell to The cBot fluidics device according to manufacturer's protocol [3] with TruSeq SR Cluster Kit v2–cBot–HS (Catalog # GD-401-2510). Operate the cBot fluidics device using the Illumina cBot software to perform the following reactions:

a. Hybridize template DNA-Hybridize template molecules onto the oligonucleotide-coated surface of the flow cell.

b. Amplify template DNA-Isothermally amplify the molecules to generate clonal DNA clusters.

c . Linearize-Chemically linearize the dsDNA clusters. This is the first step of converting dsDNA to ssDNA that is suitable for sequencing.

d. Block-Block the free 3'-OH ends of the linearized dsDNA clustersto prevent nonspecific sites from being sequenced. After this step, the flow cell is stable and can be stored.

e. Denature-Convert the dsDNA to ssDNA.

f. Hybridize sequencing primers-Hybridize a sequencing primer, or multiple sequencing primers, onto the linearized and blocked clusters. After this step, the flow cell is ready for sequencing.

Step 3. Sequencing

After the completion of the above step, the lane [lane] of the flow cell (using flow cell v1.5) is used for sequencing with four-color DNA Sequencing-By-Synthesis (SBS) technology using the illumina(R) HiSeq 2000, [machine]. The sequencing run [run] and the base call analysis are performed according to the manufacturer's protocol [4] with TruSeq SBS kit-HS (Catalog # FC-401-1001).

Step 4. Basecall

After the sequencing, sequence raw data is generated.

References/Notes:

[1] Concentration of denatured template DNA is regulated with [DNA_concentrations] as follows.

[DNA_concentrations] = 4-8 pM: 1.0 nM [DNA_concentrations] = 8-12 pM: 1.5 nM [DNA_concentrations] = 12-16 pM: 2.0 nM [DNA_concentrations] = 16-20 pM: 2.5 nM [DNA_concentrations] = 20-24 pM: 3.0 nM

[2] ' cBot_UserGuide ' [3] is described that prepare template DNA at a concentration of 10 nM using EB buffer (10 mM Tris-Cl pH 8.5) as usual protocol.

[3] ' cBot_UserGuide ' on as following URL (on 'PRODUCT SUPPORT'). File:CBot UserGuide 15006165 C.pdf http://www.illumina.com/systems/genome_analyzer/cbot.ilmn

[4] 'HiSeq 2000 User Guide' on as following URL (on 'PRODUCT SUPPORT'). File:HiSeq2000 UserGuide 15011190 D.pdf http://www.illumina.com/systems/hiseq_2000.ilmnn