Sequencing Technologies

Overview of Sequencing Technologies

Chapter Overview

• Sanger sequencing was the first major DNA sequencing technology. It is highly accurate for read lengths up to ~1kb, but is prohibitively expensive for high-throughput sequencing. Sanger sequencing is still used today, but mostly for very small sequences of DNA. Sanger sequencing is sequencing by synthesis.

• Pyrosequencing by 454 was the first next-generation sequencing technology. While it revolutionized the field, it recently became noncompetitive with Illumina technologies (see this academic paper and this report). Pyrosequencing is sequencing by synthesis.

• SOLiD sequencing is one of the first platforms NOT to sequence by synthesis. It's based on a 2-nucleotide sequencing by ligation. Since each base is passed over twice in a single run, SOLiD sequencing is very accurate. However, it has become less common, mainly due to very short read lengths (~50-75bp) and a very long time to run (2 weeks, compared to approx 24 hours for Illumina).

• Illumina sequencing generally uses short reads (50-300bp) and sequences relatively accurately with a relatively low cost, which makes it the most popular platform for most sequencing projects. Illumina sequencing is sequencing by synthesis.

• PacBio SMRT sequencing detects fluorescent labeled dNTP analogs in real time. It produces very long reads (up to 30k at once), but does so with very high error rates. SMRT sequencing is still active today, usually being used with on specific sequences such as those with low sequence complexity.

Method	Read Length	Error Rate	Cost per Gb (approximate)	Main Error Type
SOLID	50	<0.1%	$70	AT Bias
Illumina	50-300	~0.1%	$40	Substitution
454 (Pyrosequencing)	100-700	~1%	$80	Insertion/Deletion
Sanger	400-1000	<0.1%	$2,400,000	Substitution
PacBio	1000-30,000	13%	$1,000	Insertion/Deletion