OP-Illumina-sequencing-PairedEnd-v3.0
From FANTOM5_SSTAR
Protocol: OP-SOLEXA-sequencing-PairedEnd-v3.0
Author: Kaida, Kaoru
Created: Sep 17th, 2010
Update: Jul 6th, 2011
Parameters: machine, run, lane, DNA_concentrations, software, base_caller_version
Description:
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 Cluster Station fluidics device according to manufacturer's protocol [3] (Chapter 6 is skipped) with Cluster Generation Kit v4. Operate the Cluster Station fluidics device using the Illumina Cluster Station 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 is used for sequencing with four-color DNA Sequencing-By-Synthesis (SBS) technology using the illumina(R) Genome Analyzer, [machine]. The sequencing run [run] and the base call analysis are performed according to the manufacturer's protocol [4] (Chapter 4, 5 and 7 are skipped) TruSeq SBS V5-GA Kit.
Step 4. Basecall
After the sequencing, sequence raw data is generated with processing by [software] with version [base_caller_version].
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] 'Cluster Station User Guide' [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] 'Cluster Station User Guide' on as following URL (on 'PRODUCT SUPPORT').
File:ClusterStation UserGuide 15005236 B-1.pdf
http://www.illumina.com/systems/genome_analyzer/cluster_station.ilmn
[4] 'Genome Analyzer II/IIx User Guide' on as following URL (on 'PRODUCT SUPPORT').
File:GenomeAnalyzer IIx UserGuide 15015098 B.pdf
