About Next-generation
Sequencing (NGS)

Next-generation Sequencing (NGS) is a technology for determining the sequence of DNA (or RNA converted to cDNA) to study genetic variation associated with biological function or diseases. This technology, introduced for commercial use in 2005, is also called Massively-Parallel Sequencing (MPS), because it enables the sequencing of many DNA strands at the same time, while the Sanger Sequencing method by Capillary Electrophoresis (CE) can only sequence a single DNA fragment at a time. Additional advantages of NGS compared with the Sanger method include lower sample input requirements, higher accuracy, and ability to detect variants at lower allele frequencies.

An NGS workflow typically comprises three major steps, regardless of the instrument technology used:

• Library preparation
• Sequencing
• Data analysis

Library preparation ("library prep") is the first step of NGS. It allows DNA or RNA to adhere to a sequence analyzer flowcell and allows a sample to be identified. Two common methods of library preparation are ligation-based library prep and tagmentation-based library prep.  An NGS library is a collection of similarly sized DNA fragments with known adapter sequences added to both 5' and 3' ends. A library corresponds to a single sample and multiple libraries, each with their own unique adapter sequences, and can be pooled and sequenced simultaneously in a sequencing run.

NimaGen offers different targeted NGS Library Prep kits optimized for the widely adopted illumina® sequencing technology. Library preparation protocols usually consist of a multi-step process and require costly reagents and substantial hands-on-time. NimaGen EasySeq™ NGS Library Prep kits are based on proprietary Reverse Complement PCR (RC-PCR), an NGS Library Prep method aimed at providing cost-efficient and safer NGS Library Prep workflows, for both RNA and DNA sequencing.

NGS libraries require high-quality nucleic acid inputs of varying quantities, concentration, and size depending on the library preparation methods and sequencing platforms used.  Regardless of these variations, in most instances a magnetic bead-based chemistry is utilized to generate sequence-ready libraries.  Magnetic beads are typically used for NGS library clean-up and size selection, aimed at removing sequencing adaptors or PCR primers, dNTP's, enzymes, unwanted buffer components, as well as nucleic acid fragments or library molecules that are above or below a specified size range optimal for the downstream sequencing platform.

EasySeq™ NGS Library Prep by Reverse Complement PCR (RC-PCR) provides a straightforward and flexible library prep, by significantly reducing hands-on time and minimizing the risk of error and contamination versus other commercially available library prep methods:

• Pre-PCR: single-tube reaction setup for combined indexing and amplification - (c)DNA and RC-PCR Mastermix are added to a Unique Dual Indexes (UDI) plate, and loaded on to the PCR instrument
• Post-PCR: all samples are pooled to perform a single-tube NGS library clean-up, using AmpliClean™ magnetic beads
• Following standard quantification and QC, the NGS library is ready to be sequenced

Adding unique indexes to sequencing libraries allows pooling (multiplex) and sequencing multiple libraries together, saving both sequencing time and cost. 

The use of Unique Dual Indexes (UDI) plates with EasySeq™ NGS Library Prep allows increasing the number of samples sequenced per run and, when combined with robust RC-PCR, improves library prep workflow and reduces per-sample cost compared to other indexing strategies. With just a single UDI plate, 96 samples can be pooled together. 

To prevent repeated sequences in a well plate, UDI uses unique identifiers (adapters) on both ends of a sample. One UDI plate enables 96 samples to be pooled together using 96 unique Index 1 adapters and 96 unique Index 2 adapters. Each EasySeq™ assay and library type has dedicated UDI plates.

When preparing libraries for multiplexing, the use of UDI plates is recommended. With a dual-indexed library, the sequencing run includes two additional reads. These additional reads allows post-run analysis software to discriminate between individual samples with increased accuracy.