Improvements in Long Read
Automated Sequencing

Steve Roemer, Vince Boveia, Pat Humphrey, Jim Amen, and Harry Osterman
LI-COR Inc., Biotechnology Division, 4647 Superior Street, Lincoln, NE 68504

Abstract

In genomic sequencing, longer reads and higher accuracy can potentially lead to better assemblies with fewer contigs, faster finishing, fewer improperly assembled repeats, and reduced costs. Several factors were examined with the goal of increasing read length, reducing time/cost, and improving accuracy of the data.

Gel electrophoresis conditions were redeveloped for the Model 4200 (IR²) Automated DNA Analysis System by optimizing the gel matrix composition, gel thickness, buffers and electrical settings. Thinner gels and lower gel concentrations yield significant improvements in band resolution and sequencing speed for a range of gel sizes. Additionally, improvements to our base-calling algorithm have enabled analysis of previously unresolved regions of sequence ladders. The new automatic base caller generates 100-200 additional bases with increased accuracy when compared to the earlier version. The synergistic effect of these factors has produced longer read lengths with high accuracy while significantly reducing the electrophoresis run time.