OP-HiSeq2000-sequencing-cBot-v3.0

Step 1. Prepare Sample DNA for Cluster Generation

Combine the template DNA [template_DNA_concentration][1] nM and 0.1 N NaOH. Vortex briefly to mix the template solution. Centrifuge the template solution to 280 xg for one minute. Incubate for 5 minutes at room temperature to denature the template into single strands. Transfer 20 μl of denatured template to a tube containing 980 μl of pre-chilled HT1 (Hybridization Buffer) [2]. Dilute the denatured DNA to titration series of [final_DNA_concentrations][3] pM with pre-chilled hybridization buffer and dispense 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 v3–cBot–HS (Catalog # GD-401-3001). 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 v3) 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 v3-HS (Catalog # FC-401-3001).

Step 4. Basecall

After the sequencing, sequence raw data is generated.

References/Notes:

[1] 'cBot_UserGuide' [3] is described that prepare template DNA at a concentration of 2 nM (10 μl) as usual protocol.

[2] 'cBot_UserGuide' [3] is described that denature the template DNA with 0.1 N NaOH to a DNA concentration of 20 pM.

[3] Dilute the denatured DNA to the desired concentration using the following example

[final_DNA_concentrations] = 10pM : 500 ul (20 pM denatured DNA) + 500 ul (Pre-chilled HT1)

[final_DNA_concentrations] = 12pM : 600 ul (20 pM denatured DNA) + 400 ul (Pre-chilled HT1)

[final_DNA_concentrations] = 15pM : 750 ul (20 pM denatured DNA) + 250 ul (Pre-chilled HT1)

[final_DNA_concentrations] = 18pM : 900 ul (20 pM denatured DNA) + 100 ul (Pre-chilled HT1)

[final_DNA_concentrations] = 20pM : 1,000 ul (20 pM denatured DNA) + 0 ul (Pre-chilled HT1)

[3] 'cBot_UserGuide' on as following URL (on 'PRODUCT SUPPORT').

File:CBot UserGuide 15006165 F.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