Next Generation Sequencing Technologies

Practice vocabulary flashcards covering the key terms, mechanisms, specs, and instrumentation of next-generation sequencing platforms, including Illumina short-read sequencing, PacBio SMRT and HiFi technology, and Oxford Nanopore sequencing.

Pyrosequencing

An early next-generation sequencing technology introduced in 2005 that detects nucleotide incorporation through light generation.

Illumina Solexa

A short-read next-generation sequencing technology that utilizes solid-state amplification on a glass surface and reversible terminator chemistry.

Solid-State Amplification

The process in which DNA fragments are bound to the solid surface of a flow cell slide and amplified locally via bridge PCR to form clonal clusters.

Reversible Terminator Nucleotides

Chemically modified nucleotides containing a fluorescent dye and a removable blocking group on their $3'\text{-OH}$ end, allowing only a single base to be incorporated and identified per sequencing cycle.

Adapters (Illumina)

Short oligonucleotides ligated to both ends of fragmented genomic DNA that enable the single-stranded fragments to attach to complementary primers on the flow cell surface.

Bridge Amplification

A solid-phase amplification step where single-stranded DNA bends over to hybridize with adjacent surface-bound primers, forming a bridge template that DNA polymerase extends.

Denaturation (Flow Cell)

The step in Illumina cluster generation where double-stranded DNA bridges are chemically separated into single strands, leaving single-stranded templates anchored to the substrate.

DNA Cluster

A localized clonal group of several million identical double-stranded DNA molecules generated within a flow cell channel from a single original template fragment.

Sequencing by Synthesis (SBS)

A sequencing technique in which fluorescently labeled reversible terminators are added sequentially, excited by a laser, and imaged base by base as the complementary strand is synthesized.

Reference Sequence Alignment

The bioinformatics process of aligning short-read sequencing data against a known reference genome to detect single-nucleotide polymorphisms (SNPs) and unknown sequence variants.

Illumina Genome Analyzer II (GAII)

An early Illumina instrument featuring read lengths of $30\text{–}75\,\text{bp}$, a throughput of $20\,\text{Gb}$ per run across 8 lanes, run times of $1\text{–}4\,\text{days}$, and an operating cost of around $\$10,\!000$ per run.

Paired-End Sequencing Workflow

A sequencing strategy where genomic DNA is sheared into relatively short fragments ($200\text{–}500\,\text{bp}$), ligated to adapters, and sequenced from both opposite ends to determine fragment length, distance, and orientation.

Mate Pair Sequencing Workflow

A library preparation method where long DNA fragments ($2\text{–}5\,\text{kb}$) are biotinylated at their $5'$ ends, circularized, sheared to $400\text{–}600\,\text{bp}$, and affinity-purified for biotin to sequence the ends of originally distant sequences.

Proximity Mapped Reads

An Illumina method used to assess long-range genomic structures by utilizing transposases inside slide nanowells to bind, cleave, and bridge-amplify long fragments across multiple wells.

Single-Molecule Real-Time (SMRT) Sequencing

A long-read sequencing methodology developed by Pacific Biosciences where an individual, unamplified DNA polymerase anchored inside a zero-mode waveguide is monitored optically as it incorporates nucleotides in real time.

Phospholinked Hexaphosphate Nucleotides

Fluorescently labeled nucleotides utilized in PacBio sequencing that have distinct fluorophores attached to their polyphosphate chains, allowing natural cleavage and release of the label during incorporation without terminating chain elongation.

Zero-Mode Waveguide (ZMW)

A nanoscale well with a $100\,\text{nm}$ limit of detection zone and an underlying epifluorescence detector that isolates optical observation to a single anchored polymerase molecule.

SMRTbell Library

A double-stranded DNA template capped with single-stranded hairpin adapters at both ends, forming a closed circular molecule suitable for repeated rolling-circle sequencing by DNA polymerase.

Circular Consensus Sequencing (HiFi Read Generation)

A PacBio technique where DNA polymerase continuously reads around a circularized SMRTbell template across multiple passes, allowing random single-molecule errors to be corrected to yield a HiFi read with $99.9\%$ accuracy.

Sequencing Platform Accuracy

A performance metric comparing read accuracy distributions among platforms: PacBio HiFi achieves $\text{Q33} = 99.95\%$, Illumina NovaSeq achieves $\text{Q31} = 99.92\%$, and ONT Bonito achieves $\text{Q18} = 98.3\%$.

NovaSeq 6000

A modern high-throughput Illumina platform that produces $2 \times 150\,\text{bp}$ reads in a $44\,\text{hr}$ run time with an overall output of $2400\text{–}3000 \times 10^9$ bases.

Nanopore Sequencing

A direct single-molecule long-read sequencing technology that passes individual DNA strands through a tiny protein pore embedded in an electrically resistant membrane to detect characteristic disruptions in ionic current.

MinION

A compact, handheld, portable sequencing instrument developed by Oxford Nanopore Technologies that connects via USB to perform real-time single-molecule sequencing.

Ionic Current Disruption

The mechanism in nanopore sequencing where each passing nucleotide (A, T, G, or C) partially blocks the pore aperture in a base-specific manner, producing distinctive electrical current signatures that are decoded into sequence data.