A Sequel to Sanger: amplicon sequencing that scales

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• High-throughput sequencing (HTS) platforms have limitations for amplicon sequencing due to short read lengths and high error rates.
• Sanger sequencing, while accurate, is less efficient for large-scale projects.
• Existing HTS methods struggle with complex amplicons, including those with homopolymer tracts and variable GC content.

Purpose of the Study:

• To evaluate the capability of single molecule, real-time (SMRT) sequencing on the SEQUEL platform for amplicon sequencing.
• To assess SMRT sequencing's performance with diverse amplicon types, including those with GC variation and homopolymer tracts.
• To compare the cost-effectiveness and throughput of SMRT sequencing against Sanger and other second-generation platforms.

Main Methods:

• Utilized 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system.
• Tested SMRT sequencing on the SEQUEL platform with over 5000 species and 20,000 specimens.
• Analyzed amplicons with wide variations in GC composition and sequence attributes, including homopolymer tracts.

Main Results:

• SMRT sequencing demonstrated high fidelity and produced results highly similar to Sanger sequencing.
• The SEQUEL platform provided more complete amplicon coverage, particularly for sequences with homopolymer tracts.
• SMRT sequencing on the SEQUEL platform significantly reduces costs compared to Sanger and Illumina/Ion platforms, enabling analysis of 10,000 DNA extracts per run.

Conclusions:

• SMRT analysis on the SEQUEL platform generates high-fidelity sequences resilient to GC content variations and homopolymer tracts.
• The analytical costs are substantially lower than first or second-generation sequencers.
• SMRT analysis enables massive amplicon characterization, with the potential to sequence over 5 million DNA extracts annually per instrument.