A DNA sequencing strategy

A modification of Lin's systematic DNA sequencing strategy is described. A method based on the religation of compatible cohesive ends generated by Sau3AI and BamHI was developed. The original procedure has been simplified and the yield of transfectant has been greatly improved. After complete digestion with BamHI and limited cleavage with Sau3AI, the single-cut linear DNA does not have to be separated from the supercoil or the open circular DNA on an agarose gel. After ligation, the DNA is digested with the restriction enzyme between the cloning site and BamHI site again. The original intact DNA is linearized, whereas the deleted subclone is not. Therefore the background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb cDNA fragment containing the haptoglobin-related sequences. It is not necessary to purify large amounts of RF DNA (500 ng is enough) to get enough subclones. A set of subclones was produced in 1 day and the yield of plaques was about sixfold higher than that published.     

A systemic DNA sequencing strategy

A systematic DNA sequencing strategy is presented. Instead of the sequencing of randomly selected DNA fragments (the “shotgun” method), the nucleotide order is progressively determined along a DNA chain using the dideoxynucleotide termination sequencing system and the single-stranded bacteriophage vector M13 derivatives. The length of DNA along which the sequence data can be progressively read appears to be limited only by the insertion capacity of the vector. As an example of this strategy a recombinant replicative form with a 2327 nucleotide long HindIII fragment from the restriction enzyme digest of bacteriophage λ DNA was prepared. The replicative form of the recombinant was partially digested with DNAase I in the presence of Mn²⁺. As the replication origin of the phage vector was located near one end of the inserted DNA and the priming site of the vector at the other, the breaks outside the inserted DNA either destroyed the phage or removed the priming site. With the use of a unique restriction site close to the priming site, the breaks within the inserted DNA gave rise to a recombinant mixture with the inserted DNA fixed at one end and sequentially shortened at the other. Using the ddT reaction screening procedure, 11 recombinants were identified in which the inserted DNA varied in length by about 200 nucleotides. Sequencing these recombinants by the dideoxynucleotide sequencing system covered the whole 2327 nucleotides of the HindIII DNA fragment. The average number of nucleotides read from a gel was 210, which fell into the most readable region of a sequencing gel: the overlapping regions between two gels were of 33 to 48 nucleotides.     

A strategy of DNA sequencing employing computer programs.

With modern fast sequencing techniques and suitable computer programs it is now possible to sequence whole genomes without the need of restriction maps. This paper describes computer programs that can be used to order both sequence gel readings and clones. A method of coding for uncertainties in gel readings is described. These programs are available on request.     

An improved DNA sequencing strategy

A modification of Hong's systematic DNA sequencing strategy is described. The original procedure has been simplified and transfectant yield increased. After DNase I limited cleavage in the presence of Mn2+, the single-cut linear DNA does not have to be separated from supercoiled or open circular DNA on an agarose gel. After ligation, the DNA is digested with a second restriction endonuclease for which a unique cleavage site resides between the insert and the first restriction endonuclease cutting site. The original intact DNA is linearized whereas the deleted subclone is not. The background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb DNA fragment containing the araC regulatory gene from Erwinia carotovora. A set of subclones sufficient to sequence the fragment on both strands was produced in 2 days and the yield was at least 60-fold higher than in the original protocol.     

A system for shotgun DNA sequencing.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.     

Exoquence DNA sequencing.

We have developed a strategy for DNA sequencing based on exonuclease III digestion followed by double strand specific endonuclease digestion and direct dideoxynucleotide sequencing reaction. This strategy eliminates the need for subcloning, oligonucleotide primers, and prior knowledge of the DNA to be sequenced. All template and primer duplexes needed for sequencing a complete insert can be prepared in one day from uncharacterized starting DNA. Sequence information can be obtained from different regions of the DNA simultaneously. The method uses double-stranded DNA to generate single-stranded template and primer, and thus produces high quality sequence results. Commercially available dideoxy-sequencing kits are well suited for this method. The strategy should be applicable for both automatic and routine laboratory DNA sequencing.     

Partial-digest DNA sequencing.

A technique called partial-digest sequencing that permits DNA of 4-6 kb in length to be sequenced without subcloning is described. The method exploits the specific cuts introduced by partial digestion with restriction endonucleases that have 4-base recognition sites to produce ordered ladders of PCR-amplified fragments. The staggered ends contain PCR primers and can thus be individually sequenced using conventional methods to yield overlapping sequences covering the entire region. This method should have significant impact on both large and small DNA sequencing projects and find many applications in general manipulations in which ordered sets of deletions need to be produced.     

DNA sequencing and helix–coil transition. III. DNA sequencing

We show that the fine oscillatory structure of the DNA melting curve can be used to determine explicitly the nucleotide composition and the order of certain domains within the DNA. If DNA is specifically fragmented, the order of fragments can be learned directly from a comparison of the differential melting curves of the nonfragmented and fragmented DNA. The indicated information may complement exact methods of DNA sequencing. The proposed analysis is applied to bacteriophage ?X-174, whose melting curve is known. Compared to the known ?X-174 DNA sequence, the results of the analysis are found to be very accurate.     

DNA sequencing with transposons.

The use of transposons offers the possibility of a directed approach to DNA sequencing, where a target DNA up to about 6kb in length can be sequenced quickly and with minimal redundancy. Transposons are mobile DNA elements which can be inserted in a reasonably random fashion into the target DNA. An important part of this process is the location of the transposon insertions (known as mapping) and the selection of a sensible subset of transposons to use as priming sites for sequencing reactions. This paper presents a probabilistic method of scoring selected subsets of transposons and a graph-theoretic algorithm for selection of a subset of maximal score.     

Enzymatic multiplex DNA sequencing.

The problem of reading DNA sequence films has been reformulated using an easily implemented, multiplex version of enzymatic DNA sequencing. By utilizing a uniquely tagged primer for each base-specific sequencing reaction, the four reactions can be pooled and electrophoresed in a single lane. This approach has been previously proposed for use with fluorescently labelled probes (1), and is analogous to the principle used in four-dye fluorescence sequencing except that the signals are resolved following electrophoresis (2). After transfer to a nylon membrane, images are obtained separately for each of the four reactions by hybridization using oligonucleotide probes. The images can then be superimposed to reconstitute a complete sequence pattern. In this way the correction of gel distortion effects and accurate band registration are considerably simplified, as each of the four base-specific ladders require very similar corrections. The methods therefore provide the basis for a second generation of more accurate and reliable film reading programs, as well as being useful for conventional multiplex sequencing. Unlike the original multiplex protocol (3), the approach described is suitable for small projects, as multiple cloning vectors are not used. Although more than one vector can be utilized, only a library of fragments cloned into any single phage, phagemid or plasmid vector is actually required, together with a set of tagged oligonucleotide primers.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.     

A quality control algorithm for DNA sequencing projects.

Heterologous DNA sequences from rearrangements with the genomes of host cells, genomic fragments from hybrid cells, or impure tissue sources can threaten the purity of libraries that are derived from RNA or DNA. Hybridization methods can only detect contaminants from known or suspected heterologous sources, and whole library screening is technically very difficult. Detection of contaminating heterologous clones by sequence alignment is only possible when related sequences are present in a known database. We have developed a statistical test to identify heterologous sequences that is based on the differences in hexamer composition of DNA from different organisms. This test does not require that sequences similar to potential heterologous contaminants are present in the database, and can in principle detect contamination by previously unknown organisms. We have applied this test to the major public expressed sequence tag (EST) data sets to evaluate its utility as a quality control measure and a peer evaluation tool. There is detectable heterogeneity in most human and C.elegans EST data sets but it is not apparently associated with cross-species contamination. However, there is direct evidence for both yeast and bacterial sequence contamination in some public database sequences annotated as human. Results obtained with the hexamer test have been confirmed with similarity searches using sequences from the relevant data sets.     

A modified TnphoA useful for single-stranded DNA sequencing.

The TnphoA transposon constructed by Manoil and Beckwith [Proc. Natl. Acad. Sci. USA 82 (1985) 8129-8133] has been modified to permit easy isolation of single-stranded (ss) DNA of target plasmids. The intergenic region (IG) of filamentous phage f1, which consists of the phage origin of replication and packaging signal, was inserted into a nonessential region of TnphoA. This modified transposon should be useful for the analysis of genes cloned in plasmids that lack a filamentous phage IG. Transposition of TnphoA-IG into a plasmid carries the IG with it; subsequently, after infection with a filamentous helper phage, ss plasmid DNA suitable for sequence analysis and useful for oligodeoxyribonucleotide-mediated mutagenesis of TnphoA-generated fusions can be isolated. The utility of TnphoA-IG was confirmed by analysis of 'blue hops' into the bla (encoding beta-lactamase) and pspE (encoding phage shock protein) genes whose products are secreted into the Escherichia coli periplasm.     

A general coverage theory for shotgun DNA sequencing.

The classical theory of shotgun DNA sequencing accounts for neither the placement dependencies that are a fundamental consequence of the forward-reverse sequencing strategy, nor the edge effect that arises for small to moderate-sized genomic targets. These phenomena are relevant to a number of sequencing scenarios, including large-insert BAC and fosmid clones, filtered genomic libraries, and macro-nuclear chromosomes. Here, we report a model that considers these two effects and provides both the expected value of coverage and its variance. Comparison to methyl-filtered maize data shows significant improvement over classical theory. The model is used to analyze coverage performance over a range of small to moderately-sized genomic targets. We find that the read pairing effect and the edge effect interact in a non-trivial fashion. Shorter reads give superior coverage per unit sequence depth relative to longer ones. In principle, end-sequences can be optimized with respect to template insert length; however, optimal performance is unlikely to be realized in most cases because of inherent size variation in any set of targets. Conversely, single-stranded reads exhibit roughly the same coverage attributes as optimized end-reads. Although linking information is lost, single-stranded data should not pose a significant assembly liability if the target represents predominantly low-copy sequence. We also find that random sequencing should be halted at substantially lower redundancies than those now associated with larger projects. Given the enormous amount of data generated per cycle on pyro-sequencing instruments, this observation suggests devising schemes to split each run cycle between twoor more projects. This would prevent over-sequencing and would further leverage the pyrosequencing method.