Efficient low redundancy large-scale DNA sequencing at EMBL

doi:10.1016/0168-1656(95)00007-D

Journal of Biotechnology

Volume 41, Issues 2–3, 31 July 1995, Pages 121-129

https://doi.org/10.1016/0168-1656(95)00007-D Get rights and content

Abstract

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random ‘shotgun’ strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15-^∗dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

References (32)

W. Ansorge et al.
A non-radioactive automated method for DNA sequence determination
J. Biochem. Biophys. Methods
(1986)
A. Edwards et al.
Automated DNA sequencing of the human HPRT Locus
Genomics
(1990)
S. Henikoff
Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing
Gene
(1984)
S. Wiemann et al.
Simultaneous on-line DNA sequencing on both strands with two fluorescent dyes
Anal. Biochem.
(1995)
R. Wilson et al.
Nucleotide sequence analysis of the 95 kb 3′terminal region of the murine T-cell receptor a/d chain locus: strategy and methodology
Genomics
(1992)
W. Ansorge et al.
High-throughput DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory
Electrophoresis
(1992)
R. Baer et al.
DNA sequence and expression of the B95-8 Epstein-Barr virus genome
Nature
(1984)
H. Blöcker et al.
The “shortmer” appproach to nucleic acid sequence analysis I: computer simulation of sequencing projects to find economical primer sets
CABIOS
(1994)
M.S. Chee et al.
Analysis of the protein coding content of the sequence of human cytomegalovirus AD169
Curr. Top. Microbiol. Immunol.
(1990)
M. Craxton
Linear amplification sequencing, a powerful method for sequencing DNA
Methods (companion to Methods Enzymol.)
(1991)

B. Dujon et al.

The complete DNA sequence of chromosome XI of Saccharomyces cerevisiae (666 kb)

Nature

(1994)

D. Grothues et al.

Separation of up to 1000 bases on a modified A.L.F. DNA sequencer

Nucleic Acids Res.

(1993)

L.E. Kotler et al.

DNA sequencing: Modular primers assembled from a library of hexamers or pentamers

L.G. Lee et al.

DNA sequencing with dye-labelled terminators and T7 DNA polymerase: effect of dyes and dNTPs on the incorporation of dye-terminators and probability analysis of termination fragments

Nucleic Acids Res.

(1992)

J. Messing et al.

The use of single stranded phage in DNA sequencing

S. Oliver et al.

The complete DNA sequence of yeast chromosome III

Nature

(1992)

Cited by (14)

Functional genomics of smut Fungi: From genome sequencing to protein function
2014, Advances in Botanical Research
Citation Excerpt :
In a one-of-a-kind map-based cloning approach, 258 overlapping BAC clones were assembled into 28 large contigs for the 23 U. maydis chromosomes via a hybridization-based technique (Meksem et al., 2005). Subsequently, for each of the BAC clones, an ordered set of overlapping plasmid subclones was generated, again via a mapping by hybridization approach (Scholler, Benes, Voss, Ansorge, & Hoheisel, 1996; Voss et al., 1995). The resulting set of more than 45,000 overlapping plasmid clones was systematically sequenced from both ends.
In contrast to most biotrophic plant pathogens, smut fungi belonging to the family of Ustilaginaceae are facultative parasites that have a yeastlike, saprophytic phase contrasting the filamentous, parasitic growth during plant infection. This dimorphic lifestyle allows easy culturing, reverse genetic modifications and cell biological analyses in the saprophytic stage; impact on virulence or pathogenicity can then been monitored easily in infection assays. Such excellent preconditions made the maize smut fungus Ustilago maydis the most advanced model organism for fungal biotrophs. A recent key step in Ustilago research was the high-quality sequencing and manual annotation of its genome, which identified a wealth of novel virulence genes that are arranged in physical gene clusters. In next steps, the genomes of the maize head smut Sporisorium reilianum f. sp. zeae and the barley covered smut Ustilago hordei have been sequenced. These closely related pathogens exhibit significant differences regarding genome organization and gene equipment, likely reflecting different infection styles compared to U. maydis, whose unique feature is to cause tumours locally to sites of infection in vegetative tissue. Comparative analysis of these genomes as well as transcriptome profiling approaches allowed the identification of regulatory elements controlling pathogenic development as well as of secreted effector genes with important functions in plant interaction. In this chapter, we summarize the most important findings gained by genome analyses of smut fungi and define important questions that need to be addressed in the future.
Sequence analysis of genes and genomes
2000, Journal of Biotechnology
A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in its genome. The development of powerful techniques for DNA sequencing has enabled sequencing of large amounts of gene fragments and even complete genomes. Important new techniques for physical mapping, DNA sequencing and sequence analysis have been developed. To increase the throughput, automated procedures for sample preparation and new software for sequence analysis have been applied. This review describes the development of new sequencing methods and the optimisation of sequencing strategies for whole genome and cDNA analysis, as well as discusses issues regarding sequence analysis and annotation.
7 Deciphering Genomes Through Automated Large-scale Sequencing
1999, Methods in Microbiology
Purification and properties of the 5'-3' exonuclease D10A mutant of DNA polymerase I from Streptococcus pneumoniae: A new tool for DNA sequencing
1998, Journal of Biotechnology
A D10A mutation was introduced at the 5′-3′ exonuclease domain of Streptococcus pneumoniae DNA polymerase I by site directed mutagenesis of the polA gene. Introduction of the mutation resulted in a drastic decrease of the 5′-3′ exonucleolytic activity present in the wild-type enzyme. Moreover, the mutation at the D10 residue of the pneumococcal polymerase affected the dependency on metal activation of its 5′-3′ exonucleolytic activity. These results provide experimental support for the proposed direct involvement of this Asp residue in a metal-assisted 5′-3′ exonucleolytic reaction in type I-like bacterial DNA polymerases and related bacteriophage 5′-3′ exonucleases. The D10A mutant polypeptide retained the polymerase activity of its parental enzyme, it is able to incorporate correctly nucleotides in a DNA template, and efficiently uses labeled and unlabeled nucleotides analogues in DNA sequencing by the dideoxy-chain-termination method. These characteristics convert this polymerase into a useful tool for manual and automatic sequencing.
High-speed DNA sequencing in ultrathin slab gels
1997, Current Opinion in Biotechnology
There have been many recent theoretical and technical advances in the sequencing of DNA in ultrathin slab gels. These include recent experimental progress in four areas: DNA polymerases; DNA template preparation; the separation and detection of DNA bands; and base-calling algorithms. The Zimm—Lumpkin theory of electrophoresis provides a new and improved framework for understanding the operation of sequencing gels.
"Doublex" fluorescent DNA sequencing: Two independent sequences obtained simultaneously in one reaction with internal labeling and unlabeled primers
1996, Analytical Biochemistry
The novel “doublex” DNA sequencing technique that makes it possible to obtain simultaneously two independent sequences from one sequencing reaction with the use of unlabeled primers and internal labeling is described. The different sequencing products are labeled in parallel with fluorescein-15-dATP and Texas red-5-dCTP present in the same tube. The characteristics of T7 DNA polymerase are exploited to ensure that only either of the labeled dNTPs is incorporated into the corresponding sequencing products. Specificity of labeling is ensured by the selection of primers. One of the unlabeled primers is chosen to be followed by an “A,” the other by a “C” to be incorporated immediately downstream from the primer binding site. The doublex sequencing technique is applicable to the simultaneous sequencing of either the same DNA template/strand or a mixture of different templates. Combinations of unlabeled and labeled primers in the same sequencing reaction are also possible. The two sequences can be determined in parallel and on-line in the same lanes of a gel with a novel automated DNA sequencer, which was previously described for use with labeled primers.

View all citing articles on Scopus

View full text

Efficient low redundancy large-scale DNA sequencing at EMBL

Abstract

J. Biochem. Biophys. Methods

Genomics

Gene

Anal. Biochem.

Genomics

High-throughput DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory

Electrophoresis

DNA sequence and expression of the B95-8 Epstein-Barr virus genome

Nature

The “shortmer” appproach to nucleic acid sequence analysis I: computer simulation of sequencing projects to find economical primer sets

CABIOS

Analysis of the protein coding content of the sequence of human cytomegalovirus AD169

Curr. Top. Microbiol. Immunol.

Linear amplification sequencing, a powerful method for sequencing DNA

Methods (companion to Methods Enzymol.)

The complete DNA sequence of chromosome XI of Saccharomyces cerevisiae (666 kb)

Nature

Separation of up to 1000 bases on a modified A.L.F. DNA sequencer

Nucleic Acids Res.

DNA sequencing: Modular primers assembled from a library of hexamers or pentamers

DNA sequencing with dye-labelled terminators and T7 DNA polymerase: effect of dyes and dNTPs on the incorporation of dye-terminators and probability analysis of termination fragments

Nucleic Acids Res.

The use of single stranded phage in DNA sequencing

The complete DNA sequence of yeast chromosome III

Nature