High-Throughput Plasmid Purification for Capillary Sequencing

Abstract

In April 1999, the Production Sequencing Facility of the Joint Genome Institute (JGI) replaced 28 ABI 377 (Applied Biosystems) slab gel systems with 84 MegaBACE 1000 DNA capillary sequencers (Amersham Pharmacia Biotech) in the realization that a massive scaleup in DNA sequencing throughput was needed to achieve the new goals of the Human Genome Project. For our part, we were charged with the draft sequencing of human chromosomes 5, 16, and 19, encompassing ∼300 Mb or 10% of the human genome, which may contain up to 4000 genes. Our sequencing strategy was somewhat different than that of the other members of the G5 (Baylor College of Medicine, Washington University School of Medicine, Whitehead Institute, Sanger Center, and JGI). We were totally committed to using paired-end plasmid sequences, attributable in part to the documented advantages of using plasmids as opposed to M13-based sequencing vectors (Roach et al. 1995; Chissoe et al. 1997). In addition, the introduction of capillary-based DNA sequencing machines, coupled with the use of polyacrylamide as our sieving matrix, raised the throughput and uniformity requirements of our plasmid preparation. These facts required us to rethink our current DNA purification strategy.

In developing a scalable method for double-stranded plasmid template isolation, we faced the challenges of developing a high-throughput protocol that could be largely automated while maintaining the highest standards in terms of read length and pass rates. Internally, we set the bar at a 70% final pass rate with an average read length of more than 500 Phred 20 bases (Ewing and Green 1998) on lanes producing 50 or more bases. In this high-throughput environment, we could not test each and every template before DNA sequencing. Therefore, the pass rate and DNA concentration had to be robust enough to allow for limited quality control testing. In practice, the method worked so well that we abandoned our gel and fluorometric analysis to gauge pass rate and average DNA concentration.

The protocol is a combination of cell lysis and cell debris removal followed by a final polishing step to remove proteins and lysis buffer components. A key component is the use of solid phase reversible immobilization (SPRI) as our polishing step. It presented itself as a scalable and automatable method that purifies plasmids to the levels required for DNA sequencing (Hawkins et al. 1994, 1997) and PCR products (DeAngelis 1995).

Our main focus was to develop a high-throughput lysis method that uniformly cracked open the bacteria while removing substances that degrade plasmids and interfere with the SPRI process. We adopted a detergent-heat lysis method that was developed at Washington University School of Medicine (Marra et al. 1999) and modified it to meet our needs. The major optimizations were the elimination of 19 steps by using direct lysis of the cells in the bacterial media, growth in shallow well plates, and the elimination of agarose gel quality control. These changes resulted in an extremely robust process that meets the DNA quality and yield requirements for DNA sequencing with linear polyacrylamide capillary systems.

Plates are 96-well and contain pUC-18 plasmid with random 3–4 kb human DNA inserts from chromosomes 5 and 16. Pass rates are based on lanes producing >50 bases scoring Phred 20 or better. Read lengths are the average of the Phred 20-qualified bases in the passed lanes. Total samples, 960.

Acknowledgments

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