lect 21 Introduction to DNA Sequencing and Next-Generation Sequencing (NGS)

This flashcard set covers the fundamental concepts, techniques, and platforms of DNA sequencing, ranging from traditional Sanger sequencing to modern Next-Generation Sequencing (NGS) and Nanopore technologies.

DNA sequencing

The process of determining the nucleotide sequence of DNA.

Dideoxynucleotides (ddNTPs)

Molecules that resemble normal nucleotides but lack the normal $-OH$ group, possessing a $3'-H$ instead, which prevents the formation of a phosphodiester bond.

Chain termination

A process in Sanger sequencing where DNA synthesis stops whenever a labeled dideoxynucleotide is incorporated into the growing DNA strand.

Dye terminator sequencing

Another name for Dideoxy DNA sequencing, referring to the use of labeled ddNTPs to stop synthesis.

Polyacrylamide gel

A thin gel used in Sanger sequencing electrophoresis that allows for higher voltage and faster separation of DNA fragments by size.

Pyrosequencing

A "sequencing by synthesis" method developed in 1996 that detects base incorporation through the emission of light rather than chain termination.

Pyrogram

The visual result of pyrosequencing where peak height indicates whether one, two, or no nucleotides were incorporated into the new DNA strand at a specific step.

Next-generation sequencing (NGS)

High-throughput sequencing platforms, such as Illumina or Ion Torrent, that allow for massive parallel sequencing of DNA fragments.

Adapters

Universal DNA sequences ligated to target DNA fragments to simplify sample processing, allow for purification, and enable uniform library quantification and amplification.

Bridge-PCR

A solid-phase amplification process used by Illumina where single-stranded fragments form bridges on a flow cell surface to create dense clusters of double-stranded DNA.

De novo assembly

A computer-based method of assembling sequencing data into a complete sequence without using a pre-existing reference genome.

Ion Torrent Sequencing

A post-light sequencing method that detects the release of hydrogen ions ($H^+$) when a nucleotide is incorporated into DNA using a proprietary ion sensor.

Nanopore sequencing

A physical sequencing method where DNA is threaded through a microscopic pore, and bases are identified by how they perturb the flow of ions and change the electrical current.

Apyrase

An enzyme used in pyrosequencing to destroy excess deoxynucleotide precursors that were not incorporated into the DNA chain.

Reference-based assembly

The process of aligning sequencing data against a known reference genome to identify sequence differences or SNPs.