Systematic sequencing of cDNA clones using the transposon Tn5

In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inserts. We have developed a strategy for high-throughput sequencing of cDNA clones using the transposon Tn5. This approach has been tailored for implementation within an existing large-scale ‘shotgun-style’ sequencing program, although it could be readily adapted for use in virtually any sequencing environment. In addition, we have developed a modified version of our strategy that can be applied to cDNA clones with large cloning vectors, thereby overcoming a potential limitation of transposon-based approaches. Here we describe the details of our cDNA-sequencing pipeline, including a summary of the experience in sequencing more than 4200 cDNA clones to produce more than 8 million base pairs of high-accuracy cDNA sequence. These data provide both convincing evidence that the insertion of Tn5 into cDNA clones is sufficiently random for its effective use in large-scale cDNA sequencing as well as interesting insight about the sequence context preferred for insertion by Tn5.     

An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones

We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.     

Rapid verification of lambda-cDNA clones using mixed-base oligonucleotide as screening probe and sequencing primer

A rapid procedure has been developed for the isolation and verification of cDNA clones isolated from a cDNA library based on lambda vectors. Using information of the partial amino acid sequence of a protein, synthetic mixed-base oligonucleotides are first employed as a screening probe using the plaque hybridization procedure. The cDNA inserts of the clones obtained are then directly amplified by polymerase chain reaction (PCR) using primers flanking the cloning site of the vector. Besides being used for cloning into a plasmid vector, the amplified DNA's are also subjected to nucleotide sequence analysis using the same mixed-base oligonucleotides as sequencing primers. This approach allows sequencing through the region of the known amino acid sequence for direct verification of the authenticity of the clones obtained. This procedure has successfully been used for cloning and partial characterization of the gene coding for a platelet aggregation inhibitor.     

Generation of expressed sequence tags of random root cDNA clones of Brassica napus by single-run partial sequencing.

Two hundred thirty-seven expressed sequence tags (ESTs) of Brassica napus were generated by single-run partial sequencing of 197 random root cDNA clones. A computer search of these root ESTs revealed that 21 ESTs show significant similarity to the protein-coding sequences in the existing data bases, including five stress- or defense-related genes and four clones related to the genes from other kingdoms. Northern blot analysis of the 10 data base-matched cDNA clones revealed that many of the clones are expressed most abundantly in root but less abundantly in other organs. However, two clones were highly root specific. The results show that generation of the root ESTs by partial sequencing of random cDNA clones along with the expression analysis is an efficient approach to isolate genes that are functional in plant root in a large scale. We also discuss the results of the examination of cDNA libraries and sequencing methods suitable for this approach.     

Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.     

An Experimentally Derived Data Set Constructed for Testing Large-Scale DNA Sequence Assembly Algorithms

A data set consisting of DNA sequences from a large-scale shotgun DNA cloning and sequencing project has been collected and posted for public release. The purpose is to propose a standard genomic DNA sequencing data set by which various algorithms and implementations can be tested. This set of data is divided into two subsets, one containing raw DNA sequence data (1023 clones) and the other consisting of the corresponding partially refined or edited DNA sequence data (820 clones). Suggested criteria or guidelines for this data refinement are presented so that algorithms for preprocessing and screening raw sequences may be developed. Development of such preprocessing, screening, aligning, and assembling algorithms will expedite large-scale DNA sequencing projects so that the complete unambiguous consensus DNA sequences will be made available to the general research community in a quicker manner. Smaller scale routine DNA sequencing projects will also be greatly aided by such computational efforts.     

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Hierarchical shotgun sequencing remains the method of choice for assembling high‐quality reference sequences of complex plant genomes. The efficient exploitation of current high‐throughput technologies and powerful computational facilities for large‐insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole‐genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high‐quality assemblies of a large number of clones to assemble map‐based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.     

Probe-based negative selection for underrepresented phylotypes in large environmental clone libraries

Studies based on cloning and sequencing to investigate microbial diversity in a vast range of samples has become widespread in recent years. Results have revealed immense microbial diversity in many different environments, but also dominance of a few sequence types in the constructed clone libraries. Here we describe a method to enrich the clone libraries by avoiding sequencing of known, abundant sequence types, instead focusing on novel, rare ones. The protocol is based on gridding the PCR products from clone libraries on membranes and hybridisation of species-specific probes. Clones that do not give positive hybridisation results are sequenced. This method was used for fungal clone libraries from compost samples. Altogether 1536 clones were gridded and six probes used. From these clones, 59% hybridised with a probe, and therefore, only 41% of the clones were sequenced. In addition, 384 samples were sequenced to verify the hybridisation results. The numbers of false-negative (5.2%) and false-positive (3.9%) hybridisations were low. This method provides a mean of lowering the costs of sequencing projects and speeding up the process of characterising microbial diversity in environmental samples. The method is especially suitable for samples with a few dominating sequence types.     

A Modular, Positive Selection Bacterial Artificial Chromosome Vector with Multiple Cloning Sites

To construct large-insert libraries for the sequencing, mapping, and functional studies of complex genomes, we have constructed a new modular bacterial artificial chromosome (BAC) vector, pBACe3.6 (GenBank Accession No. U80929). This vector contains multiple cloning sites located within the sacB gene, allowing positive selection for recombinant clones on sucrose-containing medium. A recognition site for the PI-SceI nuclease has also been included, which permits linearization of recombinant DNA irrespective of the characteristics of the insert sequences. An attTn7 sequence present in pBACe3.6 permits retrofitting of BAC clones by Tn7-mediated insertion of desirable sequence elements into the vector portion. The ability to retrofit BAC clones will be useful for functional analysis of genes carried on the cloned inserts. The pBACe3.6 vector has been used for the construction of many genomic libraries currently serving as resources for large-scale mapping and sequencing.     

Identification of novel homologues of three low molecular weight subunits of the mitochondrial bc1 complex

Large-scale random cDNA sequencing projects have been started for several organisms and are a valuable tool for the analysis of quantitative and qualitative aspects of gene expression. However, the reliability of the obtained data is limited as most of the clones are only partially analysed on one strand. As a consequence the sequence entries derived from random cDNA sequencing projects usually comprise incomplete open reading frames. They nevertheless define complete and reliable coding sequences, if two prerequisites are fullfilled: (i) the clones encode very small proteins, and (ii) the clones have a high frequency in the cDNA-banks. The present study describes the use of cDNA databases for the identification of homologues of three low-molecular-weight subunits of the mitochondrial bc₁ complex, termed the QCR6, QCR9 and QCR10 proteins. These polypeptides are only characterized for a small number of organisms, have a scarcely defined function and exhibit a low degree of structural conservation if compared between different species. Several clones were identified for each polypeptide by searches with TBLASTN using the known sequences as probes. Most of the database entries contain complete open reading frames and sequencing queries could be excluded due to the abundancy of the clones. Multiple sequence alignments are presented for all three polypeptides and consensus sequences are given which may provide a basis for the investigation of the proteins by site-directed mutagenesis.     

用PCR直接筛选和鉴定虎纹捕鸟蛛毒素Ⅰ的重组阳性克隆

以合成的两段插入序列为上、下游引物用PCR法直接筛选插入有虎纹捕鸟蛛毒素Ⅰ（HWTX－Ⅰ）cDNA的重组阳性克隆。并用PCR法快速鉴定重组体中插入片段的正、反连接方向,扩增用引物是以位于克隆位点上游的一段载体序列上游引物,以插入序列为下游引物。对100个单克隆进行了上述两次PCR筛选鉴定,选取2个有靶片段插入并且为正向连接的重组子进行测序,其结果证实了插入片段及其方向的正确性。     

Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity.     

Sequencing of cloned DNA using bacteriophage lambda gt11 templates

A procedure for isolating and directly sequencing recombinant bacteriophage lambda gt11 DNA templates is described. Approximately 250-300 bases of sequence can be obtained directly from the lambda gt11 template, eliminating the need for subcloning prior to dideoxynucleotide sequencing of clones.     

Use of Subtractive Hybridization To Design Habitat-Based Oligonucleotide Probes for Investigation of Natural Bacterial Communities

We describe a rapid oligonucleotide probe design strategy based on subtractive hybridization which yields probes for 16S rRNA or rRNA genes of individual members of microbial communities that are specific within the context of those communities. This strategy circumvents the need to sequence many similar or identical clones of dominant members of a community. Radioactively labeled subfragments of a cloned 16S rRNA gene sequence for which a probe is required (target) were hybridized with biotinylated total 16S ribosomal DNA (rDNA) amplified from the microbial community, and the hybrids formed were subsequently discarded. The remaining enriched fragments were used to screen a library consisting of cloned subfragments of the target sequence by colony hybridization in order to identify the variable regions of the 16S rRNA gene with the required specificity. The sequencing of random clones in one 16S rDNA library demonstrated that only those clones with 100% sequence identity with the probe fragment were detected by it. Moreover, sequencing of other, randomly selected, probe-positive clones revealed 100% sequence identity with the probe. Probes developed in this way tended to correspond to more variable regions of the 16S rRNA if the target sequences were similar to the sequences of other clones in the library and to less variable regions if the target sequences were phylogenetically isolated within the clone library. Although the absolute specificity of the latter probes, as assessed by comparison with available database sequences, was lower than the absolute specificity of the probes from the more variable regions, they were specific within the context of the environmental samples from which they were derived.     

Clonal polymerase chain reaction-single-strand conformational polymorphism analysis: an effective approach for identifying cloned sequences

The amplification of target sequences from genomic DNA can result in more than one amplicon sequence being produced even when highly specific primers are used. Here we present a clonal polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) approach for screening cloned amplicons and identifying particular clones prior to sequence determination. Comparison of the PCR-SSCP patterns of the cloned amplicons with the PCR-SSCP patterns observed for the DNA templates from which the clones were derived allows PCR artifacts, different alleles, and even different loci to be differentiated prior to sequencing. Using this approach, the number of clones required for reliable sequence determination is minimized, and complex “mixed” amplicons can be resolved easily, cost-effectively, and reliably.     

Construction of a Normalized cDNA Library for the Trypanosoma cruzi Genome Project

Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.     

Nucleotide sequence analysis of 95 kb near the 3' end of the murine T-cell receptor alpha/delta chain locus: strategy and methodology.

The nucleotide sequence of a region at the 3' terminus of the murine T-cell receptor alpha/delta chain locus is presented. This region, which encodes the constant region genes for alpha and delta chain polypeptides and all 50 joining gene segments for the alpha chain polypeptide, spans 94,647 bp and includes more than 50 noncoding sequence elements important for T-cell receptor gene rearrangement and expression. DNA sequencing of this region included complete analysis of two cosmid clones and five additional restriction fragments using a random subcloning approach with various manual and automated sequencing strategies. The automated sequencing strategies hold considerable promise for future large-scale DNA sequencing efforts.