NGS Glossary: Sequencing & PCR Terms Defined

n6 technology

Who is n6?

Adaptive Amplification: An amplification workflow approach where cycling parameters are dynamically optimized per sample, rather than applied uniformly across all wells.
AutoNorm: A real-time, closed-loop amplification system that monitors fluorescence and automatically stops cycling at the optimal endpoint for each sample.
HotScan: An iconPCR feature that raises the fluorescence scan temperature of the extension step (typically 68–72 °C) to 84 °C during AutoNorm monitoring. By scanning above the melting temperature of primer dimers and other short non-specific products, HotScan dramatically reduces baseline fluorescence variability and enables more reliable detection of the true amplification signal.
icon16: A thermocycling instrument featuring 16 independently controlled wells with built-in real-time fluorescence.
icon96: A thermocycling instrument featuring 96 independently controlled wells with built-in real-time fluorescence.
iconPCR™: The world’s first intelligent thermocycler, featuring individually controlled wells designed to adjust amplification cycles in real time.

NGS basics

Adapters: Short, synthetic DNA molecules of a known sequence that are ligated to the ends of DNA or RNA fragments during library prep.
Amplicons: Copied DNA or RNA fragments that are the product of amplification.
Coverage: The average number of times a region is read during a sequencing run.
DNA Fluorescent Dye: A specialized compound that binds to DNA molecules and emits a measurable light signal (fluorescence), enabling real-time monitoring.
DNA Integrity Number (DIN): A numerical rating used to evaluate the quality of a DNA sample.
Indexes: The unique molecular barcodes attached to each sample during library prep, allowing multiple samples to be pooled together.
Low-Input / Degraded Samples: Precious biological specimens that contain damaged or scarce starting genetic material, such as cfDNA or FFPE samples.
Next Generation Sequencing (NGS): A high-throughput DNA or RNA sequencing technology.
Polymerase Chain Reaction (PCR): A technique used to make copies of a specific DNA sample exponentially.
Primers: Short single-stranded sequences that act as the starting point of DNA synthesis.
Read Depth: The total number of individual sequence reads generated for a given sample or region.
RNA Integrity Number (RIN): A numerical rating used to evaluate the quality of an RNA sample.
Sequencing: The process of reading the nucleic order (A, T, C, G) of DNA fragments or genomes.

Library prep

Amplification: The process of creating copies of a targeted DNA sequence.
Batching AutoNorm: The practice of grouping multiple biological samples for processing, amplifying, or sequencing within a single run to optimize throughput.
Traditionally a pre-amplification step — not necessary when using AutoNorm.
Library Clean Up: The process of removing unwanted reaction byproducts and surplus reagents before sequencing.
Library Preparation: The multi-step process of converting DNA or RNA samples into a collection of sequencing-ready fragments.
Normalization AutoNorm: The process of equalizing the concentration of different DNA libraries before pooling.
Traditionally a post-amplification step — done automatically during amplification by AutoNorm.
Pooling: The process of combining multiple distinct libraries so they can be sequenced together.
Quantification AutoNorm: The process of measuring the exact concentration of DNA in a sample.
Traditionally a post-amplification step — done automatically during amplification by AutoNorm.
Read Balancing: The process of adjusting library ratios so that each sample receives an equal amount of sequencing attention, preventing relatively abundant samples from dominating the sequencing output.

PCR artifacts

Over-Amplification: Occurs when a PCR run has been overcycled, which depletes reagents and leads to the exponential generation of artifacts.
Under-Amplification: Occurs when a PCR run has been terminated too early, resulting in insufficient material generated and leading to sample dropouts.
Plateau: The late stage of PCR where reaction components are exhausted, causing amplification to stall and artifacts to form.
Artifacts: Any unwanted, artificial structures or sequences created during PCR that do not accurately represent the original sample input.
Adapter Dimers: Occur when two sequencing adapters bind together, instead of attaching to a DNA insert.
Chimeras: A specific type of artifact where different DNA fragments fuse, resulting in artificial hybrid sequences.
Jackpotting: The over-amplification of random DNA fragments, leading to an uneven representation in sequencing data.
Primer Dimers: Occur when two primer molecules hybridize together, instead of the target DNA template.
Recombination: The unintended shuffling of genetic material, often caused by over-amplification.
Size Bias: The tendency of PCR to preferentially amplify shorter fragments, leading to an inaccurate representation of the original sample distribution.