Automated library construction with a fast and highly reproducible workflow
GeneRead Library Prep Kits provide an efficient and optimized workflow, generating high yields of DNA library with minimal sequence bias and low error rates. The complete library preparation workflow can be automated on the QIAcube, saving time while ensuring high reproducibility and standardization. DNA libraries are ready for use on NGS platforms from Illumina and Life Technologies.
Efficient ligation reactions and an optional, high-fidelity amplification step ensure superior library yields and quality from as low as 50 ng starting material (see figure High yields of DNA library with uniform coverage distribution
and High yields of library DNA
). The fast, one-tube procedure and optimized workflow significantly save time and effort and minimize the variability caused by handling, along with the risk of contamination (see figures Optimized workflow for Life Technologies
and Optimized workflow for Illumina
To ensure maximum yields from minimum amounts of starting material, GeneRead Library Prep Kits include an innovative, high-fidelity master mix for an optional amplification step. The unique, highly specific and processive enzyme GeneRead HiFi Polymerase, a unique provides accurate amplification of library DNA with low error rates and minimum bias (see figure Low error rates with minimal sequence bias
). With standard PCR amplification procedures, regions of DNA with high AT or GC content can result in little or no amplification, leading to misleading sequence data and NGS results. GeneRead HiFi Polymerase, together with its unique buffer formulation, ensures uniform amplification of genomic regions that contain highly variable GC content, thereby ensuring even coverage in subsequent sequencing reactions (see figure Better sequence coverage and minimal bias
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Precise size-selection of library DNA
The GeneRead Size Selection Kit uses a convenient, spin-column-based procedure to ensure precise size selection of the DNA library while effectively removing adapter dimers or monomers (see figure Precise size selection
). Automatable on the QIAcube, the easy-to-use protocol eliminates tedious handling procedures and ensures there is no risk of carryover of potentially inhibitory ethanol or beads.
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qPCR-based library quantification
One of the most important factors in an NGS experiment is accurate quantification of the prepared library to ensure quality reads and efficient data generation. Underestimation of amplifiable library molecules leads to mixed signals and non-resolvable data; conversely, overestimation results in poor yield of template-carrying beads (Ion Torrent platform) or clusters (Illumina platform) and reduced use of sequencing capacity.
The GeneRead Library Quant System uses real-time PCR to quantify NGS library molecules (see figures Targeted enrichment NGS workflow
and PCR-enabled target enrichment of genes of interest
). It specifically quantifies DNA molecules with adaptors at both ends, which are the only amplifiable molecules during emulsion PCR (Ion Torrent platform) or bridge PCR (Illumina platform) and therefore enable highly accurate quantification of amplifiable library molecules (see figure Reliable NGS library quantification
and Quantification of libraries with concentrations below the detection limit of conventional methods
). The high sensitivity of real-time PCR allows quantification of library DNA with very low concentrations, even below the detection threshold of conventional spectrophotometric methods.
The GeneRead Library Quantification System quantifies DNA after targeted PCR-enabled amplification with the GeneRead DNAseq Gene Panel System to monitor the efficiency of PCR-enrichment of targeted genes of interest with integrated controls. It can also be used for the quantification of DNA for any of the following applications:
- Whole genome
- Targeted amplification of GOIs with any other system