Enhanced throughput for infrared automated DNA sequencing

Abstract

Several enhancements have been developed and applied to infrared automated DNA sequencing resulting in significantly higher throughput. A 41 cm sequencing gel (31 cm well-to-read distance) combines high resolution of DNA sequencing fragments with optimized run times yielding two runs per day of 500 bases per sample. A 66 cm sequencing gel (56 cm wellto-read distance) produces sequence read lengths of up tol000 bases for ds and ss templates using either T7 polymerase or cycle-sequencing protocols. Using a multichannel syringe to load 64 lanes allows 16 samples (compatible with 96-well format) to be visualized for each run. The 41 cm gel configuration allows 16,000 bases per day (16 samples X 500 bases! sample X 2 ten hour runs!day) to be sequenced with the advantages of infrared technology.Enhancements to internal labeling techniques using an infrared-labeled dATP molecule (Boehringer Mannheim GmbH, Penzberg, Germany) and Sequenase (U.S. Biochemical) have also been made. The inclusion of glycerol in the sequencing reactions yields greatly improved results for some primer & template combinations. The inclusion of cz-Thio-dNTP's in the labeling reaction increases signal intensity two-to three-fold.Enhanced throughput of determining DNA sequence information by increasing the number of signal channels is an overriding objective of several groups, including our own. Signal channels can be discriminated in several ways including: geometrically (increased number of parallel channels); temporally (increased speed of data collection); optically (increased number of fluorophores); by intensity (increased number of signal amplitude states); and/or by fluorescence lifetime (via timeor phase-resolved fluorescence detection).In addition, the amount of information collected per signal channel is important. Trade-offs occur between difference approaches depending on which parameters are emphasized. For example, increasing the speed of data collection may decrease the amount of useable information. On the other hand, techniques such as pulsed field electrophoresis may succeed at increasing the information collected per signal channel at the expense of time. In addition, improvements in molecular biology techniques can increase the information content per signal channel.This report reflects results of attempts to enhance throughput by: 1) improving the information content in each lane by using longer electrophoresis gels; 2) increasing the number of gel electrophoresis lanes by progressively reducing their physical size; and, 3) improving the information content for each sample by optimizing the molecular biology of internal labeling.These enhancements exploit the advantages of near-infrared fluorescence technology, such as improved sensitivity and economy of operation. 66 !SPIE Vol. 2386 O-8194-1733-5/95/$6.OO Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/25/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx

Sequencing Technology

“…Moreover, read lengths greater than 1, OOO bases have been reported (NISHIKAWA and KAMBARA, 1992;GROTHUES et al, 1993;ZIMMERMANN et al, 1994;MIDDENDORF et al, 1995;CARRILHO et al, 1996;KLEPARNIK et al, 1996;ROEMER et al, 1997ROEMER et al, , 1998SALAS-SOLANO et al, 1998b;ZHOU et al, 2000) thus reducing the number of walking primers needed to finish a sequencing project.…”

“…1994;MIDDENDORF et al, 1995;CARRILHO et al, 1996;KLEPARNIK et al. 1996;ROEMER et al, 1997ROEMER et al, ,1998; SALAS-SOLA- ZHOU et al, 2000) as well as the use of confidence values to assess the quality of each base call (EWING et al, 1998; EWING and GREEN, 1998; RICHTERICH, 1998; RICHTE- RICH et al, 1998).…”