REPLI-g Single Cell DNA Library Kit
For DNA library construction from single cells for Illumina sequencing applications
Single cell genomic analysis enables researchers to gain novel insights across a diverse set of applications, including, developmental biology, tumor heterogeneity and disease pathogenesis and progression. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging since the amount of genomic DNA present in a single cell is very limited. PCR-based whole genome amplification methods normally have high error rates, low coverage uniformity, extensive allelic drop-outs and limited amplification yields. The new REPLI-g Single Cell DNA Library Kit leverages QIAGEN's unique multiple displacement amplification (MDA) technology to overcome these challenges by preparing a sequencing library with high fidelity and minimal bias, while retaining the sample's genomic diversity.
The REPLI-g Single Cell DNA Library Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Streamlined, one-tube library preparation in a single working dayWith the REPLI-g Single Cell DNA Library Kit, reaction setup is straightforward and handling time is greatly reduced, allowing DNA amplification and library preparation to be completed in a single working day. The kit provides a time-saving, one-tube library preparation protocol that does not require sample cleanup between steps, minimizing starting material loss and cross-contamination risk (see figure A time-saving, streamlined protocol delivers ready-to-use libraries, suitable for use on Illumina NGS platforms). Co-optimization of MDA and library construction processes enables a highly streamlined and efficient protocol, reducing MDA time to only three hours and eliminating the library amplification step. Optimized enzyme and buffer compositions ensure superior yields of high-quality, NGS-ready libraries in just one working day.
Minimal bias due to PCR-free workflowIn standard PCR amplification procedures, regions of DNA with high GC or AT content can result in little or no amplification, leading to misleading sequence data and NGS results. The REPLI-g Single Cell DNA Library Kit employs high-fidelity MDA technology to provide accurate amplification of genomes with negligible sequence bias and minimal genomic drop-outs. The REPLI-g Single Cell DNA Library Kit contains an optimized Phi29 polymerase formulation, which, together with its proprietary buffer formulation, ensures uniform amplification of genomic regions that contain highly variable GC content, thereby ensuring even coverage in subsequent sequencing reactions. Costly false-positive or -negative results are minimized with REPLI-g technology due to Phi29 polymerase, which has up to 1000-fold higher fidelity compared to normal PCR polymerases. Dedicated buffers and reagents undergo a unique, robust decontamination procedure to avoid amplification of contaminating DNA, ensuring high reliability (see figures High-quality libraries with a high percentage of mapped reads and a very low percentage of duplicates and Superior performance, greater accuracy and more even coverage).
High-quality sequencing librariesThe REPLI-g Single Cell DNA Library Kit combines the advantages of REPLI-g Single Cell technology with the ligation efficiency of GeneRead technology, delivering high-quality libraries ready for NGS, without the need for any library enrichment – avoiding additional amplification bias. Due to the high yields achieved during the WGA step, as well as the high ligation efficiency of the library construction reagents, library preparation can be performed without PCR-based library amplification, which can introduce coverage bias and reduce library diversity. The REPLI-g Single Cell DNA Library Kit allows construction of complex libraries from single cells or limited DNA materials with a high percentage of mapped reads, uniform genome coverage, high sequence complexity and low error rates, and outperforms PCR-based single cell library construction products from alternative suppliers (see figures The REPLI-g Single Cell DNA Library Kit outperforms PCR-based single cell library prep, Superior performance, greater accuracy and more even coverage and Uniform coverage over a wide range of %GC content).
The REPLI-g Single Cell DNA Library Kit includes REPLI-g sc DNA Polymerase, an optimized formulation of the innovative, high-fidelity enzyme Phi29 polymerase, to amplify complex genomic DNA using multiple displacement amplification (MDA) technology, along with gentle alkaline incubation to ensure very low DNA fragmentation or generation of abasic sites. It is specifically designed to provide high yields of amplified DNA from single cells.
The REPLI-g Single Cell DNA Library Kit uses isothermal genome amplification, termed multiple displacement amplification (MDA), which involves the binding of random hexamers to denatured DNA. This is followed by strand displacement synthesis at a constant temperature with an optimized form of the enzyme Phi29 polymerase, which has exceptionally strong strand displacement properties. Additional priming events occur on each displaced strand that serve as a template, enabling generation of high yields of amplified DNA. Phi29 polymerase, a phage derived enzyme, is a DNA polymerase with 3'→5' exonuclease activity (proofreading activity) that delivers up to 1000-fold higher fidelity compared to Taq DNA polymerase. Supported by the unique, optimized REPLI-g Single Cell buffer system, Phi29 polymerase easily solves secondary structures such as hairpin loops, thereby preventing slipping, stoppage and dissociation of the polymerase during amplification. This enables the generation of DNA fragments up to 100 kb without sequence bias.
Cell lysis and alkaline denaturation of DNA
Genomic DNA must be denatured before use in enzymatic amplification procedures, which is often accomplished using harsh methods such as incubation at elevated temperatures (heat incubation) or increased pH (chemical alkaline incubation). The REPLI-g Single Cell DNA Library Kit uses gentle alkaline incubation, allowing effective cell lysis and uniform DNA denaturation of gDNA with very low DNA fragmentation or generation of abasic sites. This results in amplified DNA with very high integrity, and maximizes the length of amplified fragments so that genomic loci and sequences are uniformly represented.
Effective elimination of detectable DNA contamination
All kit components used for WGA undergo a unique, controlled decontamination procedure to ensure elimination of all REPLI-g amplifiable contaminating DNA. Buffers and reagents are treated with an innovative and standardized procedure during manufacturing to ensure the absence of any detectable residual contaminating DNA. Following decontamination, the kits undergo stringent quality control to ensure complete functionality.
Simple, one-tube procedure – NGS-ready library prep in one day
The REPLI-g Single Cell DNA Library Kit provides a simple and reliable method to efficiently generate DNA libraries, suitable for use on Illumina NGS instruments from just a single cell or as little as picograms of DNA in 5–5.5 hours. The kit provides a complete workflow for highly uniform amplification across the entire genome, with negligible sequence bias, followed by fast, one-tube library construction (see figure A time-saving, streamlined protocol delivers ready-to-use libraries, suitable for use on Illumina NGS platforms). Dedicated buffers and reagents have been developed to deliver high yields of DNA from single cells, limited material and purified DNA, with complete sequence representation and unbiased amplification.In the first step of the WGA procedure, the cell sample is lysed and the DNA is denatured. After denaturation has been stopped by the addition of neutralization buffer, a master mix containing buffer and DNA polymerase is added. The isothermal amplification reaction proceeds for 3 hours at 30°C, and can be preprogrammed in a thermal cycler. REPLI-g SC amplified DNA can be stored long-term at –20°C with no negative effects, or used directly to generate sequencing libraries. For library construction, samples consisting of longer DNA fragments are first sheared into a random library of fragments. The median fragment sizes are dependent on the applications and sequencing read length. Following end-repair and A-addition, platform-specific adaptors, which contain sequences essential for binding the library to a flow cell for sequencing and binding sequencing primer are ligated to both ends of the DNA fragments. The WGA procedure normally results in high yields of DNA so that library preparation can be performed with a high amount of input DNA and subsequent PCR-based library enrichment can be avoided. However, if library enrichment is required, an optional, high-fidelity amplification step can also be performed that provides highly accurate amplification of library DNA with low error rates and minimum bias.
The REPLI-g Single Cell DNA Library Kit offers an efficient, PCR-free method for NGS library construction from single cells, which makes it highly suitable for applications in the fields of developmental biology, microbial research, characterizing tumor cell heterogeneity and studying the genomic diversity of circulating tumor cells.
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