DNA and RNA Sequencing Methods

Sequencing methods determine the precise order of nucleotides in DNA or, after conversion, in RNA. From Sanger's chain-termination chemistry to massively parallel next-generation platforms, these techniques let molecular pathology read genes base by base, detect mutations, and profile gene expression at scale.

Definition

DNA and RNA sequencing methods are laboratory techniques that read the linear order of nucleotide bases in a nucleic acid molecule; RNA is typically reverse-transcribed to complementary DNA before sequencing.

Scope

The topic covers first-generation (Sanger) sequencing, next-generation (massively parallel) sequencing, and RNA sequencing for transcriptome analysis, along with the general workflow of library preparation, sequencing, and read alignment. It is presented as methodological reference material rather than as clinical testing guidance.

Key concepts

• Chain-termination (Sanger) sequencing
• Next-generation / massively parallel sequencing
• Library preparation
• Sequencing reads and coverage
• Read alignment and variant calling
• RNA sequencing (transcriptomics)
• Targeted, exome, and whole-genome sequencing

Mechanisms

Sanger sequencing incorporates chain-terminating dideoxynucleotides to generate fragments of every length, which are separated by size to reconstruct the sequence (Sanger et al., 1977). Next-generation sequencing instead prepares a library of fragmented DNA, amplifies it spatially, and sequences millions of fragments in parallel, reading short stretches whose signals are assembled computationally (Margulies et al., 2005; Goodwin et al., 2016). RNA sequencing applies the same parallel approach to complementary DNA derived from RNA, quantifying transcripts and revealing splicing and expression patterns (Wang et al., 2009). Reads are aligned to a reference to identify variants or measure abundance.

Clinical relevance

Sequencing supports the detection of disease-associated variants and the molecular characterization of tumors and pathogens. This entry explains how the methods generate sequence data and is intended as a reference; it does not advise on selecting, interpreting, or acting on sequencing tests in individual patient care.

Evidence & guidelines

The methods rest on a foundational primary literature, from Sanger's 1977 chain-termination method through the first high-throughput picolitre platform (Margulies et al., 2005) and the human genome sequence (Venter et al., 2001), with later reviews surveying a decade of next-generation technologies and the rise of RNA-Seq (Goodwin et al., 2016; Wang et al., 2009).

History

Sanger's chain-termination method (1977) made DNA sequencing routine and underpinned the first sequencing of the human genome (Venter et al., 2001). The mid-2000s introduction of massively parallel sequencing dramatically lowered cost and raised throughput (Margulies et al., 2005), and within a decade next-generation sequencing and RNA-Seq had become standard tools for genomics and transcriptomics (Goodwin et al., 2016; Wang et al., 2009).

Key figures

• Frederick Sanger
• J. Craig Venter
• Marcel Margulies

Seminal works

• sanger-1977
• margulies-2005
• goodwin-2016

Frequently asked questions

How does next-generation sequencing differ from Sanger sequencing?

Sanger sequencing reads one DNA fragment at a time using chain-terminating chemistry, whereas next-generation sequencing reads millions of fragments simultaneously in a massively parallel fashion, greatly increasing throughput and lowering cost per base.

Can RNA be sequenced directly?

RNA is usually first converted to complementary DNA by reverse transcription and then sequenced; this RNA-Seq approach quantifies transcripts and reveals splicing and expression patterns.