Includes all controls.
Library Quantification Kit for Illumina® predicts library input for maximal and reliable data output in next-generation sequencing (NGS).
The quality of NGS data depends on the quality of the generated sample library. Fragmented DNA, pipetting errors, and poor PCR efficiency during target enrichment or library preparation can contribute to poor conversion rates of sample DNA into adaptor-ligated libraries. This in turn can lead to decreased sensitivity and accuracy of NGS data. The quantity of the library also determines the level of cluster density on the flow cell and directly affects the reliability of NGS results. If the cluster density is too low or too high, the depth of coverage or quality score of the data will be compromised, respectively. Since spectrophotometric and fluorometric instruments cannot distinguish the correctly adaptor-ligated, amplifiable DNA within a library, library input determined through such methods are unreliable.
Library Quantification Kit for Illumina® (LIBQ-NGS) is a qPCR assay that addresses these issues by qualifying and quantifying generated libraries intended for downstream sequencing on Illumina instruments. Using primer-probe mixes that recognize the p5 and p7 adaptor sequences, library molecules that were correctly adaptor-ligated with both oligos during library preparation are detected and quantified while incorrectly indexed molecules are left unamplified. The 6 dilution standards provided in the LIBQ-NGS kit generate a standard curve that is used to determine the amount of properly adaptor-ligated molecules within the library. Available automated analysis will provide the concentration of library molecules that can be sequenced and library input required for optimal cluster density.
After generating the library, set up reactions using reagents provided in the product IFU and run the assay on a compatible real-time PCR instrument. The assay may be completed in approximately 1 hour and 15 minutes from assay set up to results. Automated analysis is available to assist with data interpretation.
Equipment and Materials
This assay requires a real-time PCR instrument.
The kit includes:
- SYBR™ Green reaction mix.
- Primers that recognize p5 and p7 adaptor-sequences.
- 5 standards intended to generate a standard curve for determining library concentration.
The kit may be purchased separately. It is also supplied with the following kits:
- BRCA Complete (catalog no: BRCA-NGS)
The reagents in the kit are sufficient for 75 triplicate reactions including all standards, controls, and samples.