The Illumina sequencer generated raw images, utilizing sequencing control software for system control and base calling through integrated primary analysis software called RTA (real-time analysis). The BCL (base calls) binary was converted into FASTQ, utilizing Illumina package bcl2fastq.
Sequencing control software
Manufactured by Illumina
Sourced in United States
Sequencing control software is a computer program that manages and controls the operation of DNA sequencing instruments. It is responsible for coordinating the various components of the sequencing process, including data acquisition, instrument calibration, and run monitoring.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Bcl2fastq, by Illumina (1 mentions)
Pair end library preparation kit, by Illumina (1 mentions)
Hiseq sequencing kit, by Illumina (1 mentions)
S series, by Covaris (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Magmax 96 viral rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Takara rna pcr kit amv ver 3, by Takara Bio (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
4 protocols using sequencing control software
1
Illumina Sequencer Data Processing
2
Metagenomics of Tetracycline-Treated Sludge
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The metagenomic DNA extracted from the sludge cultured with 0 and 20 mg/L tetracycline was individually subjected to high-throughput sequencing using Illumina Hiseq 2000 (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The “Index 101 PE” (Paired End sequencing, 101-bp reads and 8-bp index sequence) sequencing strategy was used for the high-throughput sequencing, which generates nearly equal amount of clean reads for each sample. A base-calling pipeline (Sequencing Control Software, Illumina, San Diego, CA, USA) was applied to process the raw fluorescent images and the call sequences. The raw reads containing three or more “N” or contaminated by adapter (>15 bp overlap) were removed, and the filtered clean reads (about 1.6 Gb per each sample) were used for further metagenomic analyses. The sequencing data were deposited in the metagenomics RAST server (MG-RAST) [43 (link)] under accession number 4494851.3 (sludge treated with 20 mg/L tetracycline) and 4494856.3 (sludge without tetracycline treatment).
3
Illumina Library Preparation and Sequencing
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Genomic DNA was sheared using Covaris S series (Covaris, MS, USA). The sheared DNA was end-repaired, A-tailed, and ligated to pair-end adapters, in accordance with the manufacturer’s protocol (Pair End Library Preparation Kit, Illumina, San Diego, CA, USA). Adapter-ligated fragments were purified and dissolved in 30 μl of elution buffer, and 1 μl of the mixture was used as a template for 12 cycles of PCR amplification. The PCR product was gel-purified using the QIAquick Gel Extraction Kit (Qiagen). Library quality and concentration were determined using an Agilent 2100 BioAnalyzer (Agilent). Libraries were quantified using a SYBR green qPCR protocol on a LightCycler 480 (Roche, Indianapolis, IN, USA), in accordance with Illumina’s library quantification protocol. Based on the qPCR quantification, libraries were normalized to 2 nM, and then denatured using 0.1 N NaOH. Cluster amplification of denatured templates was performed in flow cells, in accordance with the manufacturer’s protocol (Illumina). Flow cells were paired-end sequenced on an Illumina HiSeq 2000 using HiSeq Sequencing kits. A base-calling pipeline (Sequencing Control Software (SCS), Illumina) was used to process the raw fluorescent images and the called sequences.
4
Viral RNA Extraction and Sequencing Protocol
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For each sample, 50 μl plasma was used to extract viral RNA using the MagMAX-96 viral RNA isolation kit (Ambion, Austin, TX, United States) according to the manufacturer’s instructions. The cDNA was generated with the reverse primers using the TaKaRa RNA PCR Kit (AMV) Ver.3.0 (Takara Bio, Shiga, Japan). The PCR conditions were as follows: denaturation at 94°C for 2 min; 32 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 30 s, and extension at 68°C for 30 s; and a final elongation step at 68°C for 10 min. The PCR products were run on 1% agarose gels and extracted using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). The products were quantified using a spectrophotometer (NanoDropND-1000, Thermo Fisher Scientific, Waltham, MA, United States).
The barcode-tagged products of the same fragment were pooled and purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The DNA was end-repaired, A-tailed, and PE-adapter ligated. After ligation of the adapters, each sample was purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and sequenced using the PE250 strategy on an Illumina MiSeq according to the manufacturer’s instructions. A base-calling pipeline (Sequencing Control Software, SCS; Illumina) was used to process the raw fluorescent images and the called sequences.
The barcode-tagged products of the same fragment were pooled and purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The DNA was end-repaired, A-tailed, and PE-adapter ligated. After ligation of the adapters, each sample was purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and sequenced using the PE250 strategy on an Illumina MiSeq according to the manufacturer’s instructions. A base-calling pipeline (Sequencing Control Software, SCS; Illumina) was used to process the raw fluorescent images and the called sequences.
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