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.
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.
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.
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.
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.
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.
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.
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.