| Autor: |
Schweitzer, P., Harris, A., Mandelman, D., Jackson, S., Cifuentes, F., Degoricija, L. |
| Jazyk: |
angličtina |
| Rok vydání: |
2014 |
| Předmět: |
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| Popis: |
Current methods for quantifying NGS libraries do not provide precise measurements of functionally relevant molecules. Accurate absolute quantification of NGS libraries is critical for obtaining optimal throughput from each sequencing run using the Ion Torrent PGM™ and Proton™ platforms and the Illumina HiSeq and MiSeq® platforms. Thus a need exists to precisely measure library concentration prior to clonal amplification for these sequencing systems. The QuantStudio™ 3D Digital PCR System (QS3D) offers a highly precise method for quantifying libraries prior to sequencing. Compared to alternative methods, Life Technologies chip-based dPCR approach is particularly attractive to accurately quantify libraries due to a simple workflow and eliminating the need for standard curves as required by traditional qPCR methods. TaqMan® gene expression assays were specifically designed that span the P1 and A adapters for Ion Torrent™ libraries, and that span the P5 and P7 adapters for Illumina libraries. A reaction mix was formulated by mixing diluted library with the QS3D Master Mix and TaqMan® assay and then loaded onto a QS3D chip. This chip was then sealed and the mixture amplified on a thermal cycler. Lastly, the data was visualized using the QuantStudio™ 3D AnalysisSuite Cloud Software which displays the target concentration. The concentration obtained from the digital platform was correlated to the percent of template beads (pre-enriched) for the Ion Torrent™ libraries and showed a tight range between 10–14% as determined by flow cytometry. The digital data obtained from the Illumina libraries showed a tight correlation with cluster density on an Illumina platform. We therefore present a simple and accurate workflow for quantifying NGS libraries using the QuantStudio™ 3D Digital PCR Platform. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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