From sample enrichment to genome finishing, Labgene Scientific offers a wide range of products to improve your NGS workflow:
Addressing enzyme bias
Once the library is constructed, it must be amplified in order to enrich for molecules that contain the appropriate adaptor configuration and obtain sufficient material for sequencing.
Ideally the composition/diversity of the original library and amplified library will be the same. In actuality, amplification can severely distort the representation of original library DNA – this phenomenon is the result of enzyme bias : some regions of the genome are overrepresented and other regions are underrepresented. In order to achieve sufficient coverage depth to make sense of the sequence data, the entire genome must be oversampled to ensure the sequence information for regions that are underrepresented appear at a baseline level of coverage. This increases the cost of sequencing : you have to sequence at higher coverage than you would if everything was amplified more equally.
Even with more sequencing, some regions of the genome are never represented. This results in missing sequence information.
Addressing enzyme bias>>
KAPA Library Preparation Kits provide all of the enzymes and reaction buffers required for constructing libraries from fragmented dsDNA via the following steps:
- 1. End repair: produce blunt-ended, 5’-phosphorylated fragments
- 2. A-tailing: add dAMP to the 3’-ends of the dsDNA library fragments
- 3. Adaptor ligation: ligate dsDNA adaptors with 3’-dTMP overhangs to library fragments
- 4. Amplification: PCR amplification of library fragments carrying appropriate adaptor sequences on both ends.
Targeted sample enrichment
Achieve efficient and consistent coverage of your region of interest with KAPA Long Range HotStart DNA Polymerase. High yield amplification from complex targets up to 20kb with minimal optimization.
Automated library size selection, separation and collection
The Pippin Prep and Blue Pippin are new electrophoresis platforms for preparation of size-fractionated DNA samples using pre-cast agarose gel cassettes which enable user-defined size fractions from 50bp to 50kb to be collected automatically. Product DNA is eluted into liquid buffer - no gel extraction required. The systems are especially useful for preparation of next-gen sequencing libraries, yielding higher quality DNA at a fraction of the time and labor of manual protocols.
Improved library construction for Next-Generation Sequencing
- Significant amounts of time and labor are saved compared with manual prep gels
- Extraction of narrow size ranges streamline sequencing and data analysis
- Accurate size ranges are selected with software, and reproducibly extracted from samples
- High sample recovery reduce amplification cycles and provide more unique molecules
- Minimal low molecular weight contamination improves adapter clean-up
During PCR enrichment, standard polymerases do not synthesize all library fragments with equal efficiency. This amplification bias exacerbates uneven sequence coverage. KAPA Library Amplification Kits and KAPA Real-Time PCR Library Amplification Kits have been designed to address PCR-induced bias. Kits contain the novel KAPA HiFi DNA Polymerase, engineered for high fidelity and processivity and capable of balanced amplification of complex library DNA.
- improved amplification of GC- and AT-rich genomic regions
- reduced enzyme bias resulting in improved sequencing coverage
- industry leading fidelity
The Fragment Analyzer™ Automated CE System is a fluorescence-based capillary electrophoresis instrument for both sizing and quantifying nucleic acids (DNA and RNA). By using a sensitive intercalating dye coupled with a powerful LED light source, The Fragment Analyzer™ Automated CE System obviates the need for fluorescent labeled primers and can be used to separate dsDNA fragments and RNA (mRNA and total RNA). The Fragment Analyzer™ Automated CE System is the most flexible system on the market today, with the greatest sensitivity, the highest separation resolution over a wide range, the widest dynamic range and the fastest separation times.
This instrument comes available for customers who wish to run either 12 samples or 96 samples at a time and allows to :
- quantify and qualify NGS fragments
- quantify and qualify RNA
- quantify and qualify genomic DNA
- do Mutation Detection, also known as TILLiNG, a reverse genetics technique
Accurate quantification of the number of amplifiable molecules in a fragmented library is critical to the outcome of sequencing results on next-generation sequencing platforms. Over and underestimation of library concentration result in suboptimal sequencing capacity. qPCR is widely regarded as the gold standard for accurate quantification of DNA libraries as it is the only technique capable of measuring the number of amplifiable molecules.
Accurate qPCR-based library quantification ultimately depends on three factors:
- the accuracy and reproducibility of the standards used
- the ability of the DNA polymerase used in the qPCR to amplify all adaptor-flanked molecules with equal efficiency
- accurate and reproducible liquid handling
KAPA Library Quantification Kits are rigorously tested to ensure minimal lot-to-lot variation. In addition, KAPA SYBR® FAST qPCR Kits are designed for high performance, high-throughput, real-time PCR.
KAPA Library Quantification Kit for Roche 454 FLX/Lib-A information>>
KAPA Library Quantification Kit for Roche 454 Titanium/Lib-L information>>
KAPA Library Quantification Kit for ABI SOLiD™ series information>>
KAPA Library Quantification Kit for Illumina information>>
KAPA Library Quantification Kit for Ion Torrent information>>
Delivering unsurpassed data quality for 40-cycle runs in as little as 40 minutes, the Eco™ system revolutionizes qPCR accessibility for both new and experienced Real-Time PCR users :
- High performance FAST system, high thermal uniformity
- Supports all applications including HRM and multiplexing
- Compact bench top size, easy to use software
- Ideal for Real-Time PCR NGS library amplification and quantification
Genome closure and finishing
The relatively short read lengths of NGS platforms make whole genome assembly more problematic and result in more sequence gaps than traditional Sanger sequencing methods. Without subclones, next-generation whole genome sequencing requires PCR for finishing and sequence gap closure. KAPAHiFi HotStart DNA Polymerase is recommended when finishing PCR products are sequenced using NGS. Ultra-high fidelity is important since all NGS templates are clonally amplified and are therefore more sensitive to errors introduced by PCR.
Pooled amplicon libraries for resequencing