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At least 73 records · Page 4Linked to original sources
PubMed2003

Horizontally transferred genes in plant-parasitic nematodes: a high-throughput genomic approach.

BACKGROUND: Published accounts of horizontally acquired genes in plant-parasitic nematodes have not been the result of a specific search for gene transfer per se, but rather have emerged from characterization of individual genes. We present a method for a high-throughput genome screen for horizontally acquired genes, illustrated using expressed sequence tag (EST) data from three species of root-knot nematode, Meloidogyne species. RESULTS: Our approach identified the previously postulated horizontally transferred genes and revealed six new candidates. Screening was partially dependent on sequence quality, with more candidates identified from clustered sequences than from raw EST data. Computational and experimental methods verified the horizontal gene transfer candidates as bona fide nematode genes. Phylogenetic analysis implicated rhizobial ancestors as donors of horizontally acquired genes in Meloidogyne. CONCLUSIONS: High-throughput genomic screening is an effective way to identify horizontal gene transfer candidates. Transferred genes that have undergone amelioration of nucleotide composition and codon bias have been identified using this approach. Analysis of these horizontally transferred gene candidates suggests a link between horizontally transferred genes in Meloidogyne and parasitism.

Amino Acid Sequence↗
PubMed2003

RIDOM: comprehensive and public sequence database for identification of Mycobacterium species.

BACKGROUND: Molecular identification of Mycobacterium species has two primary advantages when compared to phenotypic identification: rapid turn-around time and improved accuracy. The information content of the 5' end of the 16S ribosomal RNA gene (16S rDNA) is sufficient for identification of most bacterial species. However, reliable sequence-based identification is hampered by many faulty and some missing sequence entries in publicly accessible databases. METHODS: In order to establish an improved 16S rDNA sequence database for the identification of clinical and environmental isolates, we sequenced both strands of the 5' end of 16S rDNA (Escherichia coli positions 54 to 510) from 199 mycobacterial culture collection isolates. All validly described species (n = 89; up to March 21, 2000) and nearly all published sequevar variants were included. If the 16S rDNA sequences were not discriminatory, the internal transcribed spacer (ITS) region sequences (n = 84) were also determined. RESULTS: Using 5'-16S rDNA sequencing a total of 64 different mycobacterial species (71.9%) could be identified. With the additional input of the ITS sequence, a further 16 species or subspecies could be differentiated. Only Mycobacterium tuberculosis complex species, M. marinum/M. ulcerans and the M. avium subspecies could not be differentiated using 5'-16S rDNA or ITS sequencing. A total of 77 culture collection strain sequences, exhibiting an overlap of at least 80% and identical by strain number to the isolates used in this study, were found in the GenBank. Comparing these with our sequences revealed that an average of 4.31 nucleotide differences (SD +/- 0.57) were present. CONCLUSIONS: The data from this analysis show that it is possible to differentiate most mycobacterial species by sequence analysis of partial 16S rDNA. The high-quality sequences reported here, together with ancillary information (e.g., taxonomic, medical), are available in a public database, which is currently being expanded in the RIDOM project http://www.ridom-rdna.de), for similarity searches.

DNA, Ribosomal↗
PubMed2004

Single-tube method for nucleic acid extraction, amplification, purification, and sequencing.

BACKGROUND: The hepatitis C virus (HCV) genotype determines patient prognosis and duration of treatment, but sequencing of the gene is lengthy and labor-intensive. We used a commercially available nucleic acid extraction system to develop a single-tube extraction-to-sequencing (STETS) method for HCV genotyping. METHODS: HCV RNA was purified and amplified in tubes coated with a solid-phase matrix that irreversibly bound nucleic acid during the extraction step. After reverse transcription-PCR, the amplicon was adsorbed to the original extraction matrix for purification and use in the subsequent sequencing reactions. RESULTS: The STETS method generated genotyping-quality sequence for a range of HCV titers from 500 to 6,000,000 IU/mL. If a viral sample was detected during real-time reverse transcription-PCR, it could be sequenced and genotyped. Read lengths >600 bases were observed with the STETS method. Mixed infections were detected and genotyped if at least 15% of the minor species was present. Combining the STETS method with consecutive sequencing provided a means of performing both forward and reverse sequencing in a single tube. CONCLUSIONS: A single-tube nucleic acid extraction-to-sequencing method, which requires less time and labor than conventional methods, generates HCV sequence data that are equivalent to conventional methods and can be used to genotype HCV.

Genotype↗
PubMed2003

Gene identification through large-scale EST sequence processing.

The technology of sequencing expressed sequence tags (ESTs) offers a relatively cheap alternative to whole genome sequencing and has become a valuable resource for gene discovery. The inherent characteristics of ESTs, such as transcript redundancy, low sequence quality and high error rates, require processing of the sequences before any gene prediction can be made. The process includes EST pre-processing, analysis and similarity searches, and the data are generally stored in a database to organise the results and thereby assist the search for interesting genes.

Algorithms↗
PubMed1994

A high throughput system for the preparation of single stranded templates grown in microculture.

A high throughput system for the preparation of single stranded M13 sequencing templates is described. Supernatants from clones grown in 48-well plates are treated with a chaotropic agent to dissociate the phage coat protein. Using a semi-automated cell harvester, the free nucleic acid is bound to a glass fiber filter in the presence of chaotrope and then washed with ethanol by aspiration. Individual glass fiber discs are punched out on the cell harvester and dried briefly. The DNA samples are then eluted in water by centrifugation. The processing time from 96 microcultures to sequence quality templates is approximately 1 hr. Assuming the ability to sequence 400 bases per clone, a 0.5 megabase per day genome sequencing facility will require 6250 purified templates a week. Toward accomplishing this goal we have developed a procedure which is a modification of a method that uses a chaotropic agent and glass fiber filter (Kristensen et al., 1987). By exploiting the ability of a cell harvester to uniformly aspirate and wash 96 samples, a rapid system for high quality template preparation has been developed. Other semi-automated systems for template preparation have been developed using commercially available robotic workstations like the Biomek (Mardis and Roe, 1989). Although minimal human intervention is required, processing time is at least twice as long. Custom systems based on paramagnetic beads (Hawkins et al., 1992) produce DNA in insufficient quantity for direct sequencing and therefore require cycle sequencing. These systems require custom programing, have a fairly high initial cost and have not proven to be as fast as the method reported here.

Base Sequence↗
PubMed1999

Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels.

DNA sequencing separations have been performed in microfabricated electrophoresis channels with the goal of determining whether high-quality sequencing is feasible with these microdevices. The separation matrix, separation temperature, channel length and depth, injector size, and injection parameters were optimized. DNA fragment sizing separations demonstrated that 50-micron-deep channels provide the best sensitivity for our detection configuration. One-color sequencing separations of single-stranded M13mp18 DNA on 3% linear polyacrylamide (LPA) were used to optimize the twin-T injector size, injection conditions, and temperature. The best one-color separations were observed with a 250-micron twin-T injector, an injection time of 60 s, and a temperature of 35 degrees C. The first 500 bases appeared in 9.2 min with a resolution of > 0.5, and the separation extended to 700 bases. The best four-color sequencing separations were performed using 4% LPA, a temperature of 40 degrees C, and a 100-micron twin-T injector. These four-color runs were complete in only 20 min, could be automatically base-called using BaseFinder to over 600 bp after the primer, and were 99.4% accurate to 500 bp. These results significantly advance the quality of microchip-based electrophoretic sequencing and indicate the feasibility of performing high-speed genomic sequencing with microfabricated electrophoretic devices.

Base Sequence↗
PubMed1998

TurboPrep II: an inexpensive, high-throughput plasmid template preparation protocol.

In an effort to reduce plasmid template preparation costs for large-scale genomic sequencing projects, a boiling minipreparation protocol has been developed that enables a single individual to easily prepare 768 sequence-quality templates in 8 h, without automation. The maximum throughput for one individual using one centrifuge in the manual configuration is 1920 templates in about 8 h. The most time-consuming manual steps of this method involve pipetting, which can be automated, resulting in a significant increase in throughput and about a 60% increase in yield. This method in the fully manual configuration yields sufficient double-stranded template for two sets of cycle sequencing reactions, membrane spotting for hybridization analysis and host cell transformation for the recovery of the original recombinant. The current materials cost per template using this method is less than twenty cents. The quality of the sequence generated has been evaluated by manual 35S radioactive cycle sequencing. Initial results have shown templates prepared by this method to yield greater than 300 bp of readable sequence when the radioactively labeled products were resolved on 6% modified denaturing polyacrylamide gels.

DNA, Recombinant↗
PubMed2001

SNPing in the human genome.

More than a million genetic markers in the form of single nucleotide polymorphisms are now available for use in genotype-phenotype studies in humans. The application of new strategies for representational cloning and sequencing from genomes combined with the mining of high-quality sequence variations in clone overlaps of genomic and/or cDNA sequences has played an important role in generating this new resource. The focus of variation analysis is now shifting from the identification of new markers to their typing in populations, and novel typing strategies are rapidly emerging. Assay readouts on oligonucleotide arrays, in microtiter plates, gels, flow cytometers and mass spectrometers have all been developed, but decreasing cost and increasing throughput of DNA typing remain key if high-density genetic maps are to be applied on a large scale.

Chromosome Mapping↗
PubMed2000

Decoding the human genome sequence.

The year 2000 is marked by the production of the sequence of the human genome. A 'working draft' of high quality sequence covering 90% of the genome has been determined and a quarter is in finished form, including the first two completed chromosomes. All sequence data from the project is made freely available to the community via the Internet, for further analysis and exploitation. The challenge which lies ahead is to decipher the information. Knowledge of the human genome sequence will enable us to understand how the genetic information determines the development, structure and function of the human body. We will be able to explore how variations within our DNA sequence cause disease, how they affect our interaction with our environment and ultimately to develop new and effective ways to improve human health.

Conserved Sequence↗
PubMed2005

openSputnik--a database to ESTablish comparative plant genomics using unsaturated sequence collections.

The public expressed sequence tag collections are continually being enriched with high-quality sequences that represent an ever-expanding range of taxonomically diverse plant species. While these sequence collections provide biased insight into the populations of expressed genes available within individual species and their associated tissues, the information is conceivably of wider relevance in a comparative context. When we consider the available expressed sequence tag (EST) collections of summer 2004, most of the major plant taxonomic clades are at least superficially represented. Investigation of the five million available plant ESTs provides a wealth of information that has applications in modelling the routes of plant genome evolution and the identification of lineage-specific genes and gene families. Over four million ESTs from over 50 distinct plant species have been collated within an EST analysis pipeline called openSputnik. The ESTs were resolved down into approximately one million unigene sequences. These have been annotated using orthology-based annotation transfer from reference plant genomes and using a variety of contemporary bioinformatics methods to assign peptide, structural and functional attributes. The openSputnik database is available at http://sputnik.btk.fi.

Cluster Analysis↗
PubMed2006

Generation of a large scale repertoire of Expressed Sequence Tags (ESTs) from normalised rainbow trout cDNA libraries.

BACKGROUND: Within the framework of a genomics project on livestock species (AGENAE), we initiated a high-throughput DNA sequencing program of Expressed Sequence Tags (ESTs) in rainbow trout, Oncorhynchus mykiss. RESULTS: We constructed three cDNA libraries including one highly complex pooled-tissue library. These libraries were normalized and subtracted to reduce clone redundancy. ESTs sequences were produced, and 96,472 ESTs corresponding to high quality sequence reads were released on the international database, currently representing 42.5% of the overall sequence knowledge in this species. All these EST sequences and other publicly available ESTs in rainbow trout have been included on a publicly available Website (SIGENAE) and have been clustered into a total of 52,930 clusters of putative transcripts groups, including 24,616 singletons. 57.1% of these 52,930 clusters are represented by at least one Agenae EST and 14,343 clusters (27.1%) are only composed by Agenae ESTs. Sequence analysis also reveals that normalization and especially subtraction were effective in decreasing redundancy, and that the pooled-tissue library was representative of the initial tissue complexity. CONCLUSION: Due to present work on the construction of rainbow trout normalized cDNA libraries and their extensive sequencing, along with other large scale sequencing programs, rainbow trout is now one of the major fish models in term of EST sequences available in a public database, just after Zebrafish, Danio rerio. This information is now used for the selection of a non redundant set of clones for producing DNA micro-arrays in order to examine global gene expression.

Animals↗
PubMed2002

[Factors that influence direct sequencing of PCR products].

Factors influenced direct sequencing of PCR (polymerase chain reaction) products were investigated in this paper. It showed that the specialization of PCR products played a key role in their sequencing reactions and only which could be sequenced directly. It also showed that the PCR reaction residues (including dNTP, primers,and metal ion) affected badly on the sequencing quality, so the purification of PCR products was necessary before sequencing. In addition,the optimum templates amount in sequencing reaction rose with the increasing of their DNA size in a certain range.

English Abstract↗
PubMed1997

Ligation mediated fluorescent labeling of DNA sequencing primers.

Automated DNA sequencing utilizing fluorescently labeled primers is a proven methodology for generating quality sequence data. However, for directed primer walking strategies this necessitates synthesis and labeling of a unique primer for each sequencing reaction. Here, we describe a rapid ligation-based method of generating labeled sequencing primers. An unlabeled 5'-phosphorylated sequencing primer is ligated to a fluorescent oligonucleotide by use of a bridge primer which is complementary to portions of the previous two oligonucleotides, thus aligning them properly for ligation. The resulting fluorescent hybrid primer can be utilized directly in cycle sequencing reactions without any prior purification.

Chromosome Walking↗
PubMed2003

Sequential DEXAS: a method for obtaining DNA sequences from genomic DNA and blood in one reaction.

Sequential DEXAS (direct exponential amplification and sequencing), a one step amplification and sequencing procedure that allows accurate, inexpensive and rapid DNA sequence determination directly from genomic DNA, is described. This method relies on the simultaneous use of two DNA polymerases that differ both in their ability to incorporate dideoxynucleotides and in the time at which they are activated during the reaction. One enzyme, which incorporates deoxynucleotides and performs amplification of the target DNA sequence, is supplied in an active state whereas the other enzyme, which incorporates dideoxynucleotides and performs the sequencing reaction, is supplied in an inactive state but becomes activated by a temperature step during the thermocycling. Thus, in the initial stage of the reaction, target amplification occurs, while in the second stage the sequencing reaction takes place. We show that Sequential DEXAS yields high quality sequencing results directly from genomic DNA as well as directly from human blood without any prior isolation or purification of DNA.

Base Sequence↗
PubMed2026

Molecular Epidemiology of Non-Polio Enterovirus: Insights From L20B Cell Line Adaptation From Children With Acute Flaccid Paralysis in Pakistan.

BACKGROUND: Non-polio enteroviruses (NPEVs) are increasingly implicated in acute flaccid paralysis (AFP), often resembling poliomyelitis and complicating eradication efforts. In Pakistan, limited molecular surveillance has hindered comprehensive characterization. The L20B cell line, designed for poliovirus detection, occasionally supports NPEV replication, challenging AFP case interpretation. METHODS: Between January 2021 and December 2022, 4615 stool samples from AFP cases in children ≤15 years were analyzed. Of these, 435 were identified as NPEVs via L20B cytopathic effects and intertypic differentiation reverse transcription-polymerase chain reaction. VP1 sequencing was performed on 218 representative isolates, yielding 153 high-quality sequences (70.2%). The 224/222 primer set showed superior amplification. Phylogenetic analysis used MUSCLE alignment and the Neighbor-Joining method in MEGA X, with statistical evaluation of epidemiological data. RESULTS: NPEVs were frequently found in L20B-positive AFP cases, highlighting the cell line's limited specificity. Most cases involved children under 5, with a slight male bias. Enterovirus B was predominant (98.0%), especially Echovirus 7 (20.3%) and Echovirus 11 (10.5%), followed by Coxsackievirus B1 and Echovirus 33 (5.9% each). Geographic clustering was noted in Punjab (45.1%), Khyber Pakhtunkhwa (30.7%) and Sindh (20.3%), with seasonal peaks in late summer and early autumn. Phylogenetic data revealed localized Enterovirus B circulation with minimal genetic variation. CONCLUSIONS: The detection of diverse NPEVs in L20B-positive AFP cases emphasizes their relevance in post-polio surveillance. Incorporating routine VP1 sequencing, optimized primer use, and targeted seasonal and regional monitoring is vital to reduce diagnostic uncertainty and inform public health strategies.

Humans↗
PubMed1995

Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides.

Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.

Autoanalysis↗
PubMed1982

A left-ear advantage for identifying the emotional quality of tonal sequences.

Tonal sequences differing in emotional quality were presented dichotically. Subjects listened to a specified ear and judged the emotional tone of the stimulus heard at that ear. Accuracy was better for identifying the emotional tone of stimuli presented to the left ear. This left ear advantage was greatest where the target and competing stimuli were of different affect. The findings provide further evidence for the role of the right hemisphere in processing emotional information.

Adult↗
PubMed2003

Cell line DNA typing in forensic genetics--the necessity of reliable standards.

The incorporation of reference DNA is crucial to the validation of any DNA typing protocol. This paper aims to provide a panel of reference DNAs for actual forensic profiling strategies, i.e. autosomal and gonosomal STR typing as well as mtDNA sequencing. We have characterised three human lymphoid cell lines, GM9947, GM9948 and GM3657, and considered 58 autosomal and gonosomal microsatellites as well as the mitochondrial control region sequence. Well-established markers and STRs recently developed for forensic use were involved. K562 DNA samples which we purchased from two different suppliers were also analysed. They revealed conflicting results with regard to the ChrX STR marker genotype. Hence, we suggest that K562 is no longer used for the calibration of profiling techniques. Our investigation establishes a panel of one female and two male DNA samples as an STR allelic ladder calibration tool and offers information on six alleles of each autosome (AS) marker, three alleles of each X chromosome (ChrX) marker and two alleles of each ChrY marker. In addition, sequences of the mitochondrial control region of the three DNAs are communicated in order to provide sequencing quality control.

Cell Line, Tumor↗