Next-generation Sequencing (NGS)

Next-generation sequencing (NGS) involves three major steps: targeted NGS library preparation, including cleanup, size selection and validation, followed by subsequent sequencing and data analysis. Library preparation is crucial to the quality of NGS results and data. It ensures appropriate target DNA size and concentration, free of contaminants, with any required adapters properly ligated, making them compatible with a sequencer.

RC-PCR Powered NGS Library Prep

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 NGS is sequencing millions of fragments simultaneously (parallel), while the capillary electrophoresis (CE) based Sanger sequencing method 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.

 

The first major step in an NGS workflow, regardless of the instrument technology used, is targeted preparation of an NGS library ('library prep'), which involves subsequent multiplex PRC amplification, cleanup, size selection and validation.

 

An NGS library is a collection of similarly sized DNA fragments with known adapter sequences added to both 5' and 3' ends. A common methods of library preparation is ligation-based library prep, a process where adapters, or small known synthetic DNA sequences, are ligated to DNA fragments. 

PCR amplification is used to both increase the amount of library and enrich DNA fragments that have an adaptor ligated to each end, as the PCR primers hybridize to sequences included in the adaptors. 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. Library preparation allows DNA or cDNA to adhere to a sequence analyzer flowcell and allows a sample to be identified. 

 

Conventional amplicon-based methods for NGS library prep target enrichment involve two PCR steps: one to amplify the regions of interest, followed by a second PCR reaction to add adapter sequences (indexes) for NGS. While these methods are well established there is always a high risk of cross contamination or carry-over from first to second rounds of amplification.

 

NimaGen NGS library prep powered by Reverse Complement PCR (RC-PCR) is the only library prep method as simple as a single PCR, combining multiplex PCR amplification, adapter ligation and indexing in a closed one-tube single reaction. RC-PCR provides a straightforward but safe and robust library prep workflow with significantly reduced hands-on time and minimum risk of error and contamination versus other commercially available library prep methods. 

 

For the one-tube single multiplex reaction setup for combined indexing and amplification, sample DNA, RC-PCR Master Mix and RC-PCR Probe Panel are added to a Unique Dual Indexes (UDI) plate, and loaded on to the PCR instrument to run the RC-PCR protocol. Multiplex target amplification, sequencing adaptor addition and sample-specific unique dual indexing all occur
simultaneously in a closed-tube reaction, as simple as any normal PCR!

Unique Dual-Indexed Sequencing

Indexed sequencing is a method that allows multiple libraries to be pooled and sequenced together from just a single plate, saving both sequencing time and cost. Indexing libraries requires the addition of a unique identifier, or index sequence, to DNA samples during library preparation. When preparing libraries for multiplexing, the use of Unique Dual Indexes (UDI) plates is recommended. Dual indexes increase the accuracy of sample identification by adding another identifier to minimize issues caused by index-hopping events. Dual-indexed libraries allow higher multiplexing and the ability to run more samples per lane.

 

With a dual-indexed library, the sequencing run includes two additional reads, called the Index 1 Read and Index 2 Read. These additional reads allows post-run analysis software to discriminate between individual samples with increased accuracy. To prevent repeated sequences in the wells of a plate, Unique Dual-Indexed sequencing 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 (i7) adapters and 96 unique Index 2 (i5) adapters. 

 

The use of universal Unique Dual Indexes (UDI) plates with EasySeq™ or IDseek® 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. 

NGS Library Cleanup and Size Selection

Following multiplex RC-PCR amplification, samples are pooled to perform a single-tube NGS library cleanup and size selection, mostly using magnetic beads. Following standard quantification and QC, the NGS library is ready to be sequenced.

 

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 (AmpliClean™ or AMPure XP) is utilized to generate sequencing ready libraries. Magnetic beads are typically used for NGS library cleanup 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.

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