A Guide to the NGS Workflow

The advent of capillary instrumentation and the use of fluorescent-based detection methods, accompanied by automated analysis, has moved traditional DNA Sequencing into ‘Next Generation’ Sequencing (NGS). NGS, now also known as high-throughput sequencing, generates and analyzes millions of sequences per run, allowing researchers to sequence, resequence and compare data at a rate previously not possible. With ever-increasing capabilities of contemporary sequencers as well as developments in sample preparation, a variety of useful and specialized applications are now available to academic, industrial and clinical laboratories.

NGS Sample Preparation Workflow:

The following are some general considerations in preparing libraries for next gen sequencing.

DNA Fragmentation: The library fragment size depends mainly on the desired insert size (between the adaptors) and the limitations of the NGS platform. Illumina’s cluster amplification step following adapter ligation can accommodate a range of up to 1500 bp. For Ion Torrent, fragment sizes of 100 to 600 bp should be suitable. Commercial kits are available for enzymatic fragmentation that specify one sequencing platform and detail fragmentation size outputs.

Adapter Ligation: Commercial enzyme kits for adapter ligation contain reagents tailored to the sequencing platform. The general workflow involves end repair of the DNA fragments followed by ligation of platform-specific adaptors. The major difference between Illumina and Ion Torrent is that the latter uses blunt-end ligation. Kits typically include all the enzymes (such as ligases and polymerases) and buffers necessary, and some feature additional barcodes for multiplexing.

Library Amplification: For either Illumina or Ion Torrent platforms, many commercial library preparation kits include PCR polymerases for subsequent amplification following adapter ligation. Some feature high-fidelity and hot-start polymerases for improved coverage and lower duplication rates.

cDNA Synthesis: Kits are available for RNA sequencing applications that include reagents for reverse transcription into cDNA, either by PCR or PCR-free. Some also feature enrichment for specific RNA types, either by capturing mRNA or depleting rRNA. These allow for streamlined library construction directly from RNA samples ranging from inputs of 25 to 1000 ng.

Target Enrichment: The large amount of data generated by whole genome sequencing can complicate data processing and analysis. As a workaround, portions of genomes may be enriched to focus on key genes. With target enrichment, DNA segments can be enriched either by hybridization-based capture or multiplex PCR. Several kits are available for the in-solution capture using biotinylated RNA or oligonucleotide probes that bind to streptavidin beads.

Quantification: Prior to the sequencing run, sequencing libraries require accurate quantification to ensure good data output and quality. NGS quantification kits are available that utilize qPCR, which are selective for the molecules with the right adaptor sequences. For convenience and consistency, kits include complete sets of reagents and some feature prediluted DNA standards.

Next Gen Sequencing Platforms:

“Sequencing by synthesis” is perhaps the most well-established sequencing method, and is used by the 454, Illumina, Qiagen, and Ion Torrent (Thermo Fisher) platforms, with each platform utilizing their own proprietary technologies. Instrument models within a platform may come in varying levels of sequencing capabilities and throughput. Sample loading chips and kits for a given instrument may also be scalable to feature additional higher-throughput options. Be sure to keep the application in mind when making a selection. Whole-genome or whole-transcriptome sequencing may require higher throughputs, and de novo sequencing and metagenomic sequencing may benefit from longer read lengths. Use our Next Gen Sequencers search tool to easily compare side-by-side specifications of the latest NGS instruments on the market.

Next Gen Sequencing Kits and Reagents:

At the disposal of researchers are a variety of kits and reagents that can help streamline and speed up procedures in the NGS workflow. Next gen sequencing kits contain ready-to-use sets of reagents, such as primers, oligos, adapters, beads, buffers, and more. Another advantage of kits is that they are optimized, quality-controlled, and with minimal lot-to-lot variability. These tools can help ensure that each NGS experiment will be consistent and easy to initiate. Browse our catalog of products from trusted suppliers in next gen sequencing.