Successful sequencing requires high-quality library construction of sufficient yield, where the workflow depends on the NGS application. This includes a series of complex steps: Input sample QC, DNA or RNA library construction and target enrichment (optional), library amplification, and library quantification prior to sequencing.
Because of the number of steps involved and their inherent complexities, each step can potentially introduce errors or biases that could significantly impact the success and reliability of sequencing results. Therefore, the use of high-quality reagents and optimized protocols are required to obtain superior sequencing libraries with the lowest bias, and the highest and most uniform coverage.
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Roche offers a range of sample preparation reagents and high-quality enzymes engineered through our Directed Evolution technology for every step of the NGS sample preparation workflow.
DNA: Library Preparation
DNA library preparation for Next-Generation Sequencing (NGS) involves fragmentation of target DNA molecules into varying sizes, end-repair & A-tailing, and ligation of platform-specific adapters to the library. The libraries are then amplified and cleaned up subsequently. Depending on the nature of the DNA sample (e.g. fresh sample versus formalin-fixed paraffin embedded (FFPE) tissue), the workflow may require optimization.
Fragmentation of DNA can be achieved through mechanical or enzymatic shearing. While mechanical shearing is considered the gold standard, enzyme-based fragmentation affords speed, less hands-on time, and convenience to enable automation of the workflow. Since the adapter size is constant, the library size is adjusted based on the desired insert size and the type of application.
The KAPA Library Preparation Kits include modules required for end-repair, A-tailing, ligation, and amplification. KAPA HiFi DNA polymerase has been optimized to reduce bias during amplification to ensure uniformity of coverage across the genome. With KAPA Adapters and Cleanup Beads, Roche offers a complete library preparation solution for NGS.
Benefits of the KAPA Library Preparation Kits
- Compatibility with a wide variety of DNA inputs and sample types offering high yields of adapter-ligated library molecules
- Reduced PCR amplification bias resulting in improved sequencing coverage
- Flexible kit formats to allow for PCR-free workflows
- High-quality library construction reagents supplied in convenient master-mix formats
- Kits are compatible with the Illumina sequencing platform and provide qualified automated methods.
KAPA HyperPrep Kits offer a streamlined mechanical shearing library preparation protocol, combining several enzymatic steps and eliminating bead cleanups to 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 solutions with KAPA Adapters* and KAPA Pure Beads*.
KAPA HyperPlus Kits, our most advanced library preparation option, provide a streamlined workflow that includes fully automatable fragmentation and library preparation in a single tube. This integrated solution combines enzymatic fragmentation, with the quality of mechanical shearing, and the speed and convenience of tagmentation-based workflows.
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KAPA EvoPlus is our newest kit on the block and will make you rethink your perception of enzymatic fragmentation. It combines the speed and efficiency of enzymatic fragmentation with the exceptional performance and robustness of mechanical shearing, taking the best of both worlds to form the new gold standard in library preparation. With a simplified workflow, automation-friendly plated ready mixes, and less time-consuming prep steps, labs can now focus their resources elsewhere whilst having the peace of mind about the quality of results. All that comes with the usual outstanding single vendor support across the entire workflow. KAPA HyperPrep Kits offer complete library preparation solutions with KAPA Adapters* and KAPA (Hyper)Pure Beads*.
* These marked products are also available as stand-alone products.
RNA: Library Preparation
RNA sequencing (RNA-Seq) leverages Next-Generation Sequencing (NGS) to provide insights into global transcriptional events, coding mRNA transcripts or 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 offer workflows for different sample types and RNA-Seq applications that are proven, simple and complete. This portfolio of KAPA RNA HyperPrep Kits contain high-quality enzymes, including KAPA HiFi DNA polymerase, developed through our Directed Evolution Technology for constructing RNA libraries with minimal GC bias and uniform sequence coverage.
The KAPA RNA HyperPrep Kits utilize a novel chemistry to enable the combination of enzymatic steps and fewer reaction purifications. This results 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. It is compatible with mRNA capture as well as ribosomal depletion.
Benefits of KAPA RNA HyperPrep Kits
- Single-day library construction for Illumina sequencing, inclusive of RNA enrichment
- Robust performance across different sample types and input amounts
- Higher success rates with low-input and degraded samples
- Integrated service and support for the entire workflow from RNA to sequencing-ready library
- Validated KAPA RNA HyperPrep Kit data analysis solutions provided through the Genialis platform
KAPA RNA HyperPrep Kits: Specifications
Library amplification
Most next-generation sequencing (NGS) library construction workflows include one or more PCR amplification steps to add functional elements (e.g., sample indices, molecular barcodes or flow cell oligo binding sites), enrich for sequencing-competent DNA fragments and/or generate a sufficient amount of library material for downstream processing. PCR is also applied for other purposes in NGS workflows. Whenever PCR is performed, care must be taken to select appropriate enzyme and protocol parameters, to ensure high efficiency, minimize amplification bias and limit the introduction of sequencing artifacts.
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Roche Sequencing Solutions offers a family of KAPA HiFi Kits containing KAPA HiFi DNA Polymerase, engineered using Directed Evolution technology. The KAPA HiFi enzyme contributes to higher overall sequencing quality and economy by offering high efficiency, low bias amplification of DNA and cDNA for both NGS and PCR applications. This translates to more uniform coverage of difficult regions and lower duplication rates allowing researchers to unlock the potential of their samples. In addition, the KAPA HiFi family includes KAPA HiFi Uracil+ Kits, which contain a uracil-tolerant variant, KAPA HiFi Uracil+ DNA Polymerase. This enzyme still offers the same benefits enabled by KAPA HiFi, but is also tolerant to amplification of modified nucleic acids, including bisulfite-converted DNA.
Benefits of KAPA HiFi Kits
- Higher and more uniform coverage with lower drop out of difficult regions
- Higher yields, low duplication rates and fewer wasted sequencing reads
- Higher success rates with different sample types/applications
- Convenience with the consistency of one core enzyme
- Trusted as shown through over 10 years in the industry as well as thousands of peer-reviewed publications
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 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. It 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. 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.
KAPA Unique Dual-Indexed Adapters are high-quality, ready-to-use, full-length adapters for ligation-based library construction in Illumina® sequencing workflows. KAPA Unique Dual-Indexed (UDI) Adapters comprise 96 adapters with non-redundant index combinations to mitigate the impact of index misalignment. Roche Universal Blocking Oligo Kits eliminate the need for adapter-matched blocking oligos, affording a simple and faster workflow.
As part of the new and validated KAPA HyperCap (v3) workflow, the KAPA Universal Adapter is a short adapter with T-overhangs that ligates to library fragments coming out of the A-tailing step of the workflow. The standard version carries no indexes, while the KAPA Universal UMl Adapter includes unique molecular identifiers that improve correct duplicate filtering and thereby increase sensitivity, especially in low-input applications like somatic oncology.
When using KAPA Universal Adapters, sample barcodes have to be incorporated in the following library amplification step by using KAPA UDI Primer Mixes. These primer mixes include forward and reverse primers and carry unique 8-nucleotide indices that allow for library pooling before target capture and sequencing.
KAPA Universal Adapters together with KAPA UDI Primer Mixes replace KAPA UDI Adapters in version 3 of the HyperCap workflow.
KAPA Adapters selection guide:
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