BioE 5411 Lecture 10: DNA Sequencing Technologies and Methods

Comprehensive vocabulary flashcards covering the principles, generations, chemistry, workflow, and data analysis of DNA sequencing technologies based on BioE 5411 Lecture 10.

Identity (in DNA sequencing)

The sequencing readout that determines what specific nucleotide sequence is present in a sample.

Variation (in DNA sequencing)

The sequencing readout that identifies which specific bases or structural features differ relative to a reference or between samples.

Abundance (in DNA sequencing)

The quantitative sequencing readout that measures how many molecules or copies of a specific sequence are present.

Heterogeneity (in DNA sequencing)

The sequencing readout that identifies which individual cells or distinct molecules differ across a mixed population.

First-Generation Sequencing

Sequencing technologies—such as Sanger chain-termination and Maxam-Gilbert sequencing—that infer base identity using labeled nucleotides and electrophoresis, generating fragment read lengths of $500\text{--}1000\,\text{bp}$.

Second-Generation Sequencing (NGS)

High-throughput short-read sequencing platforms (such as 454, Solexa, Ion Torrent, and Illumina) achieved through the massive parallelization of sequencing reactions, generating reads of $\sim 50\text{--}500\,\text{bp}$.

Third-Generation Sequencing

Long-read sequencing platforms (such as PacBio and Oxford Nanopore) that read native DNA molecules in real time with single-molecule resolution, yielding reads averaging tens of kilobases ($\text{kb}$).

Deoxynucleotide triphosphate (dNTP)

A standard nucleotide containing a $3'\text{-OH}$ group, which enables DNA polymerase to continue extension because the $3'$ hydroxyl can attack the phosphate of an incoming nucleotide.

Dideoxynucleotide triphosphate (ddNTP)

A modified chain-terminating nucleotide lacking a $3'\text{-OH}$ group, preventing subsequent phosphodiester bond formation and halting chain extension by DNA polymerase.

Clean trace (Sanger chromatogram)

A high-quality Sanger sequencing electropherogram characterized by sharp, well-separated peaks with one dominant base call at each nucleotide position.

Mixed base (Sanger chromatogram)

A Sanger chromatogram signal where two distinct color peaks overlap at the same position, indicating a heterozygous single nucleotide polymorphism (SNP) or a mixed template.

Poor trace (Sanger chromatogram)

A low-quality Sanger sequencing readout characterized by overlapping, poorly resolved, or low-intensity baseline-noise peaks that should not be overinterpreted.

Bacterial Artificial Chromosome (BAC)

An engineered DNA cloning vector derived from bacterial plasmids used to propagate large genomic inserts in Escherichia coli, playing a foundational role in the Human Genome Project.

Whole-Genome Shotgun Sequencing

A sequencing strategy in which multiple copies of an entire genome are sheared randomly into variable-sized fragments, sequenced without prior physical mapping, and assembled computationally based on overlapping sequence segments.

Hierarchical Shotgun Sequencing

A genome sequencing strategy where the genome is first divided and mapped into large segments of known order (such as BAC clones), followed by shearing each known segment into smaller pieces for sequencing and automated assembly.

Sequencing Coverage

A statistical measure of average sequencing depth across a genome, calculated approximately as $\text{Coverage} \approx \frac{\text{number of reads} \times \text{read length}}{\text{genome length}}$.

Pyrosequencing

A bioluminescent sequencing-by-synthesis method that detects nucleotide incorporation by measuring light produced by a multi-enzyme cascade involving DNA polymerase, ATP sulfurylase, luciferase, and apyrase upon inorganic pyrophosphate ($\text{PP}_i$) release.

ATP Sulfurylase

An enzyme in pyrosequencing that quantitatively converts adenosine $5'\text{-phosphosulfate}$ ($\text{APS}$) and released inorganic pyrophosphate ($\text{PP}_i$) into $\text{ATP}$.

Luciferase (in pyrosequencing)

An enzyme that utilizes $\text{ATP}$ to convert luciferin into light, creating a detectable luminescent peak proportional to the amount of incorporated nucleotides.

Apyrase

A nucleotide-degrading enzyme in pyrosequencing that continuously degrades unincorporated $\text{dNTPs}$ and excess $\text{ATP}$ between reaction cycles.

NGS Library Preparation

The process of preparing genomic DNA for high-throughput sequencing by fragmenting the gDNA sample and ligating specialized adapters to both ends of each fragment.

Bridge Amplification

An isothermal PCR method in Illumina flow cells where single-stranded DNA fragments bend over to hybridize to surface-bound complementary primers, forming bridge structures that amplify into dense, clonal clusters.

Sequencing by Synthesis (SBS)

A next-generation sequencing approach in which DNA polymerase incorporates fluorescent, reversible-terminator nucleotides one base per cycle, followed by imaging and cleavage of the dye and blocking group to reset for subsequent cycles.

Reversible Terminator

A nucleotide analogue featuring a cleavable fluorescent dye and a chemical blocking group at the $3'$ position that allows only a single base addition per cycle until intentionally removed.

Library Multiplexing

The pooling of multiple distinct DNA sequencing libraries into a single run by tagging each library's fragments with unique sample-specific barcode/index sequences.

Demultiplexing

The post-sequencing bioinformatic sorting step that assigns reads back to their corresponding original samples using their unique barcode or index sequence.

Cell Barcode

A unique synthetic oligonucleotide sequence assigned to all transcripts originating from an isolated single cell during single-cell RNA sequencing (scRNA-seq).

Unique Molecular Identifier (UMI)

A short, distinct molecular tag appended to individual RNA/cDNA molecules prior to amplification to accurately count original transcripts and correct for PCR amplification bias.

Trajectory Inference (Pseudotime Analysis)

A bioinformatic analysis technique used in single-cell RNA sequencing to computationally model continuous dynamic biological processes, lineages, and cell differentiation states over time.