Next generation sequencing (NGS), also known as massively parallel sequencing, is a revolutionary technique in the life sciences, making it possible to sequence entire genomes in a fraction of the time needed for other methods. By reading millions or even billions of short DNA or RNA fragments in parallel, NGS technologies allow for higher throughput in shorter runtimes and help ensure full coverage and accuracy in sequencing.
In addition to whole genome sequencing (WGS) for research or clinical purposes, NGS can be used for whole exome sequencing (WES), targeted sequencing of specific genome regions of interest, transcriptome profiling through RNA sequencing (RNA-Seq), epigenetic and metagenomic analyses, and more. NGS allows life science researchers to study genetic variation, discover new species, investigate pathogens such as viruses and bacteria, and better understand how our genes impact our daily lives and our health.
The typical NGS workflow consists of three main steps (after nucleic acid extraction and purification) – library construction, sequencing, and data analysis. Among these steps, library construction is critical as it prepares the nucleic acid sample to attach to the flow cell and be sequenced in a massively parallel fashion. Without this step, the sample would not be compatible with the NGS instrumentation, and would not allow for the short reads that give the technique its characteristic speed and throughput.
NGS library preparation involves fragmentation of the DNA or RNA to be sequenced, followed by end repair and the attachment of adapters to both ends of the fragments; these adapters are oligonucleotides that are complementary to those on the flow cell. Fragmentation can be completed mechanically, such as through sonication or nebulization, or enzymatically, often with the use of restriction endonucleases. The adapters used for library construction must be compatible with the flow cell being used, and these adapters can also include barcodes, also called indexes, to identify a specific sample, allowing multiple samples to be sequenced in one run (i.e. multiplexing). Once a library of fragments with adapters is created, the library can be quantified using methods such as fluorometric spectroscopy or real time PCR (rT-PCR), and amplified via PCR if necessary. Additional purification steps, such as fragment size selection, can also be performed to improve the quality of the prepared library.1
Optimizing your NGS libraries sets the stage for successful downstream sequencing and data analysis, and streamlining your library preparation allows you to take full advantage of speed and throughput capabilities of the NGS method. There are now many kits available that aim to make NGS library preparation faster and easier, combining the necessary reagents and components with user-friendly or automatable workflows. Choosing the best kit for your lab will depend on your specific needs, instruments and applications; below is a roundup of some currently available NGS library preparation solutions to help you get started.
Products for NGS Library Preparation
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Illumina’s DNA PrepKit is designed to allow fast and flexible sample preparation for WGS, with an estimated prep time of about 3.5 hours. The kit allows library prep to be automated using liquid-handling robots and supports a wide DNA input range of 1-500 ng for small genomes, or 100-500 ng for large genomes. The use of Illumina’s bead-linked transposome chemistry in this kit reduces steps by integrating fragmentation, normalization and addition of sequencing primers into one step, and also increases yield of properly-sized fragments. This kit also includes an extraction workflow for blood, saliva, dried blood spots or bacterial colonies, and is compatible with Illumina NGS systems.
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Zymo Research’s Zymo-Seq RiboFree Total RNA Library Kit allows RNA libraries to be constructed in a little as 3.5 hours and is designed to ensure thorough depletion of unwanted ribosomal RNA (rRNA) in any sample from any species. The probe-free chemistry used in this kit reduces off-target removal of non-ribosomal RNA to as little as 1.8%, with rRNA depletion as high as 90% or more. The kit is automation-friendly, comes with SPRI beads and pre-mixed reagents, supports an RNA input range from 100 ng to 5 µg and is compatible with Illumina sequencing systems.
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Collibri PCR-free PS DNA Library Prep Kits by Invitrogen, a part of Thermo Fisher Scientific, are designed for library construction of non-amplified, physically sheared DNA fragments. This kit offers high coverage for inputs as low as 500 ng, providing confidence in SNP and INDEL calling for challenging samples. The protocol for this kit can be completed in under two hours, which includes end conversion, adaptor ligation and cleanup steps. Visual feedback of preparation progress is provided at each step via inert dyes that change the color of reaction mixes as components are added, and the process is also amenable to the use of liquid-handling robots and other conventional automation. This kit is recommended for WGS, resequencing and de novo assembly applications, and is compatible with Illumina sequencing systems.
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NEXTFLEX DNA-Seq Kits by PerkinElmer feature enhanced adapter ligation technology designed to increase the number of unique sequencing reads available in the prepared library. These kits can be used for DNA sequencing (DNA-Seq) or chromatin immunoprecipitation sequencing (ChIP-Seq) and enable a simplified workflow for end repair, ligation, gel-based cleanup and PCR amplification of pre-fragmented DNA. The 96-well plate compatible volumes supported by these kits allows for workflow steps to be automated and the process from end repair to amplification can be completed in under 4 hours. These kits support genomic DNA inputs of 10 ng, 100 ng or 1 µg and are compatible with Ion Torrent sequencing systems.
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The NEBNext Fast DNA Fragmentation & Library Prep Set by New England BioLabs features a simple, streamlined workflow, including enzymatic fragmentation, that can be completed in about 2 hours with minimal hands-on time. The set is compatible with inputs of 10 ng to 1 µg of DNA and fragmentation can be tuned for insert size, with minimal sample loss. This set includes the adapters and primers for singleplex libraries but are compatible with some third-party barcode adapters for multiplexing applications. New England BioLabs also offers a version of the kit without enzymatic fragmentation for mechanically sheared DNA fragments, and both sets are compatible with Ion Torrent sequencing systems.
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The SEQuoia Complete Stranded RNA Library Prep Kit by Bio-Rad allows for the construction of RNA libraries that are >99% stranded, with capture of long and short RNAs in a single library, even from limited or low-quality samples. The kit features Bio-Rad’s unique SEQzyme chemistry that integrates cDNA synthesis with adapter addition in a continuous reaction to capture all types and size of RNA species, improving the diversity and quality of prepared libraries. The protocol for this kit minimizes pipetting steps and can be completed in less than 4 hours, and supports an RNA input from 100 pg to 1 μg. The kit is also integrated with Bio-Rad’s SEQSense Analysis Solutions and Toolkit for bioinformatics and is compatible with Illumina sequencing systems.
References
- "Next-Generation Sequencing Illumina Workflow - 4 Key Steps," Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/next-generation-sequencing/illumina-workflow.html