NGS Library Preparation
Library preparation is the first step of next generation sequencing. Before DNA or RNA samples can be sequenced, nucleic acids must be isolated, fragmented, end-repaired, and covalently linked to adapters using ligation or tagmentation methods. The complexity of a well-prepared NGS library should fully and accurately reflect the complexity of the sample, but will also reflect any biases introduced during library preparation. A key goal in preparing a DNA or RNA library for next generation sequencing is to maximize complexity while reducing PCR or other amplification-introduced biases, as the quality of the resulting library can strongly impact NGS results.
We offer a portfolio of NGS library preparation tools designed to simplify workflows and facilitate the preparation of high quality DNA or RNA libraries that accurately represent sample complexity while reducing bias for whole genome sequencing (WGS), whole transcriptome analysis (WTA), total RNA sequencing, and miRNA and small RNA sequencing applications.
Related Product Resources
Genomic DNA Extraction Kits
For DNA library preparation, high molecular weight genomic DNA (gDNA) needs to be extracted from cells, tissues, or other sample types. Purified genomic DNA should have minimal shearing and biological and chemical contaminants for optimal performance in subsequent amplification or sequencing reactions.
The Fire Monkey™/Fire Flower™ high molecular weight DNA extraction kit enables isolation of high purity genomic DNA with minimal shearing and low molecular weight nucleic acid contaminants.
- Fire Monkey™ is a standard spin-column process that extracts high molecular weight DNA with average strand lengths of >100kb from either bacterial or mammalian cells in 1 hour.
- Fire Flower™ is a standard spin-column process that can size select extracted DNA from any sample source within 15 minutes. Fire Flower™ can increase the overall average strand length by 30% while depleting DNA fragments up to 30kb.
Whole Genome Amplification Kits
DNA sequence analysis is often limited by small amounts of available sample or low extraction yield. When limited starting material is attainable, amplification techniques can be used to increase the amount of starting DNA. Whole genome amplification (WGA) can be used to pre-amplify both intact and highly fragmented DNA samples for input into NGS workflows.
The SeqPlex-I WGA kit allows amplification of small quantities of DNA or degraded or highly fragmented DNA for direct input onto Illumina® next-generation sequencing (NGS) flow cells.
- Facilitates sequencing from as little as 100 pg of DNA
- Enhanced primers for complete genome coverage, minimal sequence bias, and amplicon size ideal for next generation sequencing (NGS)
- Cost-effective: No additional NGS library prep step
- Compatible with Illumina® next-generation sequencing
The SeqPlex Enhanced DNA Amplification Kit for whole genome amplification is designed to facilitate next-generation sequencing from extremely small quantities or from degraded/highly fragmented DNA. This kit has been developed to integrate into the Illumina®, SOLiD™, or 454 sequencing workflows.
- Random priming technology amplifies fragmented DNA such as ChIP or FFPE
- Facilitates sequencing from as little as 100 pg of ChIP DNA
- Enhanced primers for complete genome coverage, minimal sequence bias, and primer removal
- Compatible with Illumina®, SOLiD™, or 454 library prep for next generation sequencing
Whole Transcriptome Amplification Kits
The main goal in preparing an NGS library for RNA-Seq is to maximize transcriptome library complexity to fully represent the starting pool of sequences while reducing bias. RNA-Seq for high-throughput gene expression profiling and transcriptome analysis is commonly challenged by low quantities of starting RNA. Whole transcriptome amplification (WTA) can be used to generate sufficient amounts of sequencing targets from small amounts of RNA but requires high-fidelity transcript replication without loss or distortion of specific mRNAs to reduce library bias.
The SeqPlex-I WTA kit allows amplification of small quantities of reverse transcribed RNA or degraded RNA for direct input onto Illumina® next-generation sequencing (NGS) flow cells.
- Amplifies fragmented or extremely small quantities of total RNA. Fragmented or intact RNA from all sources including FFPE and RIP are easily amplified using random priming technology.
- Semi-degenerate library primer design ensures more complete transcriptome coverage and efficient priming
- Fewer Steps: No need to fragment cDNA before sequencing
- High-efficiency: Amplifies ds-cDNA in 8 hours or less
- Cost-effective: No longer requires an additional NGS library prep step
- Compatible with Illumina® next generation sequencing
The SeqPlex RNA Amplification Kit for whole transcriptome amplification (WTA) is designed to facilitate next-generation sequencing (NGS) from small quantities or from degraded/highly fragmented RNA (e.g. RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples). The SeqPlex kit allows the user to pre-amplify RNA samples for input into an NGS workflow.
- Random priming technology amplifies low quantities of fragmented or intact RNA from all sources including FFPE and RIP.
- Semi-degenerate library primer design for more complete transcriptome coverage and efficient priming.
- No need to fragment DNA before sequencing.
- Amplifies ds-cDNA in 8 hours or less.
- Compatible with all next generation sequencing platforms except Pacific Bioscience.
Small RNA Library Preparation
NGS technologies have revolutionized the study of small RNAs including miRNA, siRNA, and piRNA that have been shown to play a vital role in post-translational regulation of gene expression. Small RNA sequencing has been an increasingly popular approach in small RNA discovery and profiling. However, small RNA library preparation methods can introduce significant bias, specifically during adapter ligation steps.
The RealSeq®-AC RNA library preparation kit offers a novel method for preparing miRNA and small-RNA sequencing libraries that reduces incorporation bias in NGS. By using a novel single adapter and circularization, RealSeq® greatly reduces library preparation bias. This technology solves the problem of commonly used sequencing library preparations that lead to underdetection of many miRNAs.
- Significantly reduces sequencing bias in small-RNA sequencing
- Accurately quantifies biologically relevant small RNAs
- Allows for more efficient discovery of novel small RNAs
Library Cleanup
NGS library cleanup involves the removal of sequencing adaptors, PCR primers, dNTPs, enzymes, and buffer contaminants while enabling selection of nucleic acids that are in the correct size range for downstream sequencing. Library cleanup methods should maximize yield and recovery, minimize the presence of biological and chemical contaminants, and remove undesired nucleic acid fragment or library molecules in preparation for sequencing.
The HighPrep™ PCR Clean-up System is a paramagnetic bead-based post-PCR clean-up reagent designed for efficient purification of DNA fragments and for size-specific selection of amplicons. The purification consists of removal of salts, primers, primer-dimers, dNTPs, as DNA fragments are selectively bound to the magnetic bead particles. The magnetic beads are used for different library prep chemistries for NGS.
- High recovery of >100 bp amplicons
- Adaptable to high throughput liquid handling systems
- Stable recovery of amplicons > 100bp, predictable and consistent size selection
- No centrifugation or filtration leads to higher and purer yields
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