Next generation sequencing (NGS) technologies provide a high throughput means to generate a large amount of sequence data. However, quality control (QC) of sequence data generated from these technologies is extremely important for meaningful downstream analysis.
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Sample Quality Control
The North Texas Genome Center starts an NGS experiment with high-quality DNA or RNA samples. We rely on spectrophotometric (Nanodrop), fluorometric (Qubit) and gel electrophoretic methods (Agilent 4200 TapeStation) to QC starting material. Agilent’s RNA Integrity Number (RIN) must be more than 7.
Library Quality Control
Most NGS libraries are checked on the Agilent Bioanalyzer before sequencing. This verifies the insert size is as expected and that there are no contaminating adapter-dimers. These can be a major issue on the clustering chemistry for NovaSeq 6000, iSeq and MiSeq sequencing platforms.
Sequencing Quality Control
There are several commonly used sequencing QC metrics. Sequence Analysis Viewer (SAV) and FASTQC analysis reports for each sequencing lane.
Sequence Analysis View (SAV)
SAV is an Illumina tool used to monitor sequencing during or check QC after a run. View the user guide.
Equipment
Our facility is equipped with state-of-the-art instrumentation for quality control.
Agilent 4200 TapeStation
The Agilent 4200 TapeStation system is an established automated electrophoresis tool for DNA and RNA sample quality control. Fully automated sample processing enables the unattended analysis of size, concentration, and integrity. Choose any sample number between 1 and 96 and analyze at constant cost per sample. The TapeStation system provides a complete solution for true end-to-end sample quality control within any next-generation sequencing (NGS) or Biobank workflow. This covers the full DNA and RNA range and offers high levels of flexibility in a simplified workflow with ready-to-use Screen Tapes.
Invitrogen Qubit 4 Fluorometer
The Invitrogen Qubit 4 Fluorometer is the next generation of the popular benchtop fluorometer designed to accurately measure DNA, RNA, and protein quantity. The new Qubit 4 also easily measures RNA integrity and quality. The easy-to-use touch screen menus make it easy to select and run the assays you need, with results displayed in just a few seconds.
Questions?
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