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Library construction is a critical step in the next generation sequencing (NGS) workflow. DNA or RNA samples are first fragmented to the appropriate size and then sequencing platform-specific adapters are added. In most cases, libraries have to be amplified before the next step in the process. Each step can potentially introduce errors or biases that could significantly impact library quality and ultimately affect the quality and reliability of sequencing results. It is therefore imperative to use the highest quality reagents and optimized protocols to obtain the best sequencing libraries, lowest bias, and the highest and most uniform coverage.
KAPA Library Quantification Kits contain all the reagents needed for the accurate, reliable and reproducible qPCR-based quantification of next-generation sequencing (NGS) libraries prepared for sequencing on Illumina and IonTorrent platforms. Kits include KAPA SYBR FAST qPCR Master Mix (formulated with different passive reference dyes for different qPCR instruments), a platform-specific library quantification primer premix, and a pre-diluted set of DNA standards.
Six pre-diluted DNA Standards and appropriately diluted NGS libraries are amplified using platform-specific qPCR primers that target adapter sequences. The average Cq value for each DNA Standard is plotted against its known concentration to generate a standard curve. The standard curve is used to convert the average Cq values for diluted libraries to concentration, from which the working concentration of each library is calculated.
Preparation of a DNA library for next generation sequencing (NGS) platforms entails several steps that have a direct influence on the quality of the sequencing library, and ultimately on the reliability of sequencing results. Roche Sequencing Solutions offers best-in-class* DNA library preparation kits containing high-quality enzymes and reaction buffers developed through our Directed Evolution Technology. The KAPA Library Preparation Kits include modules required for end repair, A-tailing, ligation, and amplification (for Illumina platforms) and offer complete library preparation solutions with KAPA Adapters and KAPA Pure Beads.
KAPA HyperPlus Kits, the most advanced library preparation option from Roche, provide a streamlined workflow that includes fully automatable fragmentation and library preparation in a single tube. Building on industry-leading library construction efficiencies, this integrated solution combines enzymatic fragmentation, similar in quality to mechanical shearing, with the speed and convenience of tagmentation-based workflows. KAPA HyperPlus Kits offer complete library preparation solution with KAPA Adapters and KAPA Pure Beads. The kits are compatible with the Illumina sequencing platform and provide qualified automation methods.
KAPA HyperPrep Kits offer a streamlined library preparation protocol that combines several enzymatic steps and eliminates bead cleanups to significantly reduce library preparation time and improve consistency. The novel, single-tube chemistry offers further improvements to library construction efficiency, particularly for challenging samples such as FFPE and cell-free DNA. KAPA HyperPrep Kits offer complete library preparation solution with KAPA Adapters and KAPA Pure Beads.
Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer
KAPA HTP/LTP Library Preparation Kits contain optimally formulated enzymes selected through our directed evolution technology and implement a highly optimized "with bead" strategy that enable higher library construction efficiency and facilitate automation. Kits offer higher success rates than conventional library construction kits — particularly from lower inputs and challenging samples such as FFPE and ChIP DNA — in both high- and low-throughput sample preparation pipelines. KAPA HTP/LTP Library Preparation Kits provide a complete library preparation solution with KAPA Adapters and KAPA Pure Beads.
Spatial genomic heterogeneity within localized, multifocal prostate cancer
Library preparation for next-generation sequencing (NGS) often involves an amplification step for obtaining fragments enriched for adapter ligated ends and for increasing the library size, both of which are essential for effective detection of sequences during a sequencing run. However, PCR can often introduce bias for sample composition, leading to potential underrepresentation of these fragments in a sequencing read.
Roche Sequencing Solutions offers KAPA Library Amplification Kits, which contain KAPA HiFi DNA polymerase, a novel enzyme engineered through our directed evolution technology, for ultra-high fidelity and robustness. KAPA HiFi improves the overall sequencing quality with low amplification bias, more uniform coverage of difficult regions, lower duplication rates compared to other options, and amplifies NGS libraries with industry-leading fidelity.
KAPA Library Amplification Kits include KAPA HiFi DNA Polymerase, a novel enzyme engineered using our directed evolution technology for ultra-high fidelity and robustness. KAPA HiFi has become the enzyme of choice for next-generation sequencing (NGS) library amplification due to its ability to amplify complex DNA populations with high fidelity, high efficiency and very low amplification bias. This results in lower duplication rates and improved coverage of GC- and AT-rich regions, promoters, low-complexity and other challenging regions.
KAPA HiFi Uracil+ DNA Polymerase is a modified version of KAPA HiFi DNA Polymerase that is engineered to tolerate uracil residues in bisulfite-treated DNA. Bisulfite treatment of DNA results in the conversion of unmethylated cytosine residues into uracil, while the methylated residues are left unmodified. Enzymes used for library amplification in bisulfite-sequencing workflows must be able to read through uracil residues and tolerate low concentrations of AT-rich DNA. Traditional high-fidelity DNA polymerases are typically not suitable for this type of application as the enzymes stall when a uracil is encountered. KAPA HiFi Uracil+ DNA Polymerase has been developed to read through uracil residues while still retaining the performance benefits of HiFi DNA Polymerase, such as high yields, low-bias, and uniform sequencing coverage.
RNA sequencing (RNA-Seq) leverages next generation sequencing (NGS) to provide insights into global transcriptional events, coding mRNA transcripts or on the profiles of RNA species, such as lncRNA. RNA library preparation for NGS utilizes specific strategies, such as enrichment of mRNA or depletion of rRNA, based on the objective of the sequencing experiment.
Roche Sequencing Solutions offers a portfolio of products containing high-quality enzymes developed through our Directed Evolution Technology for constructing RNA libraries with minimal GC bias and even sequence coverage. KAPA RNA Library Preparation Kits include KAPA HiFi DNA Polymerase, which provides industry-leading fidelity. Roche Sequencing Solutions provides a complete library preparation solution with KAPA Adapters and KAPA Pure Beads. These kits are designed to be automation-friendly on a majority of commercially available liquid handlers.
The KAPA RNA HyperPrep Kits utilize a novel chemistry that enables the combination of enzymatic steps and fewer reaction purifications, resulting in a truly streamlined solution for the preparation of high-quality RNA-seq libraries. The strand-specific workflow is flexible, supporting library construction from lower-input amounts and degraded samples and is compatible with mRNA capture and ribosomal depletion. KAPA RNA HyperPrep Kits contain all reagents required for RNA library preparation, with the exception of KAPA Adapters (available separately).
KAPA mRNA HyperPrep Kits enrich mRNA in samples and provide a focused view of the protein-coding regions in the transcriptome. mRNA capture beads are used prior to library preparation with the KAPA RNA HyperPrep workflow. The kits enrich for mRNA over non-polyadenylated species such as ribosomal, precursor and noncoding RNAs.
The KAPA RNA HyperPrep Kit with RiboErase (HMR) targets and depletes rRNA using DNA probes and RNase H to provide improved coverage of transcripts of interest, including precursor mRNAs and important regulatory species, such as noncoding RNAs. Sequencing of rRNA-depleted total RNA samples provides a more comprehensive representation of the whole transcriptome. This kit is suitable for total RNA input from human, mouse and rat species only.
The KAPA Stranded RNA-Seq Kit combines the use of a "with bead" protocol with KAPA HiFi DNA Polymerase selected through our directed evolution technology for high efficiency, industry-leading fidelity and low-bias amplification to provide superior quality RNA libraries with flexible input amounts. Roche Sequencing Solutions provides a complete library preparation solution with KAPA Adapters and KAPA Pure Beads, available separately.
The KAPA Stranded mRNA-Seq Kit makes use of KAPA mRNA Capture Beads prior to library preparation to enrich for mRNA over non-polyadenylated species such as ribosomal, precursor or noncoding RNAs. The kits contain KAPA HiFi DNA Polymerase, selected through our directed evolution technology for high-efficiency and low-bias library amplification and are optimized for the improved coverage of GC-rich and low-abundance transcripts. Roche Sequencing Solutions provides a complete library preparation solution with KAPA Adapters and KAPA Pure Beads, available separately.
Benefits
The KAPA Stranded RNA-Seq Kit with RiboErase (HMR) targets and depletes rRNA from various sample types (human, mouse, rat) and provides more consistent and effective rRNA depletion than traditional bead-based capture methods. Efficient rRNA depletion prior to library preparation results in increased coverage of transcripts of interest, including noncoding and precursor transcripts. This kit is suitable for total RNA input from human, mouse and rat species only. Roche Sequencing Solutions provides a complete library preparation solution with KAPA Adapters and KAPA Pure Beads, available separately.
Library preparation for next generation sequencing (NGS) involves several steps – fragmentation of nucleic acids to required lengths, end repair and ligation of adapters to ends of target sequences, library amplification and quantification. A critical step in DNA and RNA library construction is to select for DNA fragments of appropriate size and to remove adapter dimers. Adapter quality and bead performance can have a profound impact on the outcome of library construction, particularly when working with low-input and challenging samples. Roche Sequencing Solutions offers high-quality KAPA Pure Beads and KAPA Adapters that are functionally tested for NGS library construction. They are supplied as stand-alone products to support flexibility.
KAPA Pure Beads offer a tunable and highly consistent solution for reaction purification and size selection in DNA and RNA next-generation sequencing (NGS) library construction workflows.
Compatible with all KAPA library preparation kits for DNA and RNA sequencing, the high-quality KAPA Single-Indexed and Dual-Indexed Adapters can be used with Illumina library construction protocols employing standard adapters. Compatible with the HyperCap Workflow v2.0.