Characterisation of the Novel HLA-B*27:05:64 Allele by Sequencing-Based Typing.
HLA-B*27:05:64 differs from HLA-B*27:05:02:05 by one nucleotide substitution in codon -21 in exon 1.
Humans↗
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Results for “Base Sequence”
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Characterisation of the Novel HLA-B*44:357:02 Allele by Sequencing-Based Typing.
HLA-B*44:357:02 differs from HLA-B*44:357:01 by one nucleotide substitution in codon 160 in exon 3.
Humans↗
Characterisation of the Novel HLA-C*07:1184 Allele by Sequencing-Based Typing.
HLA-C*07:1184 differs from HLA-C*07:01:01:01 by one nucleotide substitution in codon-18 in exon 1.
Humans↗
Characterisation of a Novel HLA-A*02:558 Allele by Sequencing-Based Typing in a Chinese Individual.
HLA-A*02:558 differs from HLA-A*02:06:01:01 by one nucleotide substitution in codon 96 in exon 3.
Humans↗
Characterisation of the Novel HLA-DPB1*1784:01 Allele by Sequencing-Based Typing.
HLA-DPB1*1784:01 differs from HLA-DPB1*514:01 by one nucleotide substitution in codon 57 in exon 2.
Humans↗
Base sequence alterations induced by ultraviolet radiation in the URA3 locus in yeast.
Explore the source record for details and available documents.
Amino Acid Sequence↗
An automated film reader for DNA sequencing based on homomorphic deconvolution.
An automated reader for electrophoresis based DNA sequencing methods is described that provides fast and accurate sequence determination. Digitized sequencing lanes are processed with homomorphic blind deconvolution in preparation for peak detection, interlane alignment, peak refinement and base calling. Initial reads from direct blot sequencing films have error rates of about 1% at the rate of 5 nucleotides/s. Typical read lengths are 500-600 nucleotides. The described reader is a significant improvement over existing readers and could be an essential component in the sequencing efforts of the Human Genome Project.
Base Sequence↗
Microbial DNA typing by automated repetitive-sequence-based PCR.
Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of Neisseria meningitidis infections. N. meningitidis outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.
Automation↗
DNA fingerprinting of Candida rugosa via repetitive sequence-based PCR.
A repetitive sequence-based PCR (rep-PCR) technique was developed to characterize the genotypic relatedness among Candida rugosa isolates. Two repetitive sequences, viz., Care-2 and Com29 from Candida albicans, were used to design primers Ca-21, Ca-22, and Com-21, respectively. When used alone or in combination, these primers generated discriminatory fingerprints by amplifying the adjacent variable regions of the genome. Twenty-three isolates from burn patients, eight from other human sources, and four C. rugosa isolates pathogenic in animals were placed into nine fingerprinting groups. Different primers placed these isolates into identical groups, indicating that rep-PCR is a specific and reproducible technique for molecular characterization of C. rugosa. Moreover, these primers unequivocally discriminated among other important Candida species such as C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, and C. lusitaniae. These data confirm the conservation of repetitive sequences in Candida species. Because of its ease and sensitivity, rep-PCR offers a relatively rapid and discriminatory method for molecular typing of C. rugosa in outbreaks.
Animals↗
Identification of four novel HLA-DOA alleles, DOA*010106, DOA*0102, DOA*0103, and DOA*0104N, by sequence-based typing*.
DOA sequences are currently known to have identical protein sequences. However, in this study, we report four novel allele types of human leucocyte antigen-DOA, including one synonymous and three non- synonymous amino acid changes from the Korean population. DOA*010106 has identical protein sequence with previously known DOA*010103 except one nucleotide difference at codon 45 (TCG-->TCA). In contrast, DOA*0102 and DOA*0103 have a sequence change at codon 99 (CTG-->GTG) and codon 105 (CGC-->TGC), causing non-synonymous amino acid changes, Leu99Val and Arg105Cys, respectively. In addition, DOA*0104N has a sequence deletion at codon 36 (CCC-->CC-), resulting in a frame shift leading to a stop codon at codon 62.
Alleles↗
Newly Developed Structure-Based Methods Do Not Outperform Standard Sequence-Based Methods for Large-Scale Phylogenomics.
Recent developments in protein structure prediction have allowed the use of this previously limited source of information at genome-wide scales. It has been proposed that the use of structural information may offer advantages over sequences in phylogenetic reconstruction, due to their slower rate of evolution and direct correlation to function. Here, we examined how recently developed methods for structure-based homology search and tree reconstruction compare with current state-of-the-art sequence-based methods in reconstructing genome-wide collections of gene phylogenies (i.e. phylomes). While structure-based methods can be useful in specific scenarios, we found that their current performance does not justify using the newly developed structure-based methods as a default choice in large-scale phylogenetic studies. On the one hand, the best performing sequence-based tree reconstruction methods still outperform structure-based methods for this task. On the other hand, structure-based homology detection methods provide larger lists of candidate homologs, as previously reported. However, this comes at the expense of missing hits identified by sequence-based methods, as well as providing sets of homolog candidates with higher fractions of false positives. These insights help to guide the use of structural data in comparative genomics and highlight the need to continue improving structure-based approaches. Our pipeline is fully reproducible and has been implemented in a Snakemake workflow. This will facilitate a continuous assessment of future improvements of structure-based tools in the AlphaFold era.
Phylogeny↗
Sequence-based diagnosis of tyrosinase-related oculocutaneous albinism: successful sequence analysis of the tyrosinase gene from blood spots dried on filter paper.
BACKGROUND: A large number of mutations of the tyrosinase gene result in oculocutaneous albinism (OCA). Therefore, at present, sequence analysis of the tyrosinase gene has become necessary to diagnose OCA patients and their relatives. OBJECTIVE: The aim of this study was to facilitate the sequence-based diagnosis of tyrosinase-related OCA by using small amounts of the patient's blood. METHODS: Blood spots dried on filter papers were used as sources of genomic DNA. The exons and flanking regions of the tyrosinase gene were amplified by polymerase chain reaction (PCR) and were directly sequenced in both directions. RESULTS: We successfully amplified all exons of the tyrosinase gene by PCR and were able to characterize compound heterozygous mutations of R278X and + delta C310 in the patient's gene. CONCLUSION: Recent advances of PCR-related technology allowed us to use fairly limited samples of blood for sequence analysis of the tyrosinase gene.
Albinism, Oculocutaneous↗
HLA-C high resolution typing: analysis of exons 2 and 3 by sequence based typing and detection of polymorphisms in exons 1-5 by sequence specific primers.
HLA-C high resolution sequence based typing developed in this study involves a unique DNA amplification encompassing exon 1 to intron 3 and four fluorescent sequencing reactions covering exon 2 and 3. Both dye primer and dye terminator sequencing techniques were performed and results compared. This approach allowed the identification of all of the 50 HLA-C allelic variants so far described, except for two allele pairs that are distinguished by non-coding nucleotide changes (Cw*12021 = 12022, Cw*15051 = 15052) and three allele pairs (Cw*0701 = 706, Cw*1701 = 1702 and Cw*1801 = 1802) that share the same nucleotide sequence in exon 2 and 3. For complete subtyping of these allelic variants, an amplification based on sequence specific primers (PCR-SSP) was used. No ambiguous heterozygous combinations of alleles were detected in our panel so far. HLA-C typing data obtained by this method were compared with data from serological and low resolution PCR-SSP typing, which had been performed previously on the samples sequenced.
Base Sequence↗
Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes.
Previously described primers for PCR amplification of variable immunoglobulin (Ig) genes were based on gene sequences. To include the large number of amino acid sequences of antibodies whose DNA has not been sequenced and to ensure a maximal fit to rearranged human Ig variable region genes, we have made a comprehensive comparison of both protein and nucleotide sequences. The resulting set of 15 primers was able to amplify a wide range of rearranged antibody variable region genes. Restriction sites included in the primers facilitate cloning of the PCR products into various expression vectors. Sequence analyses of PCR-amplified cDNA derived from a polyclonal B cell population showed that maximal enrichment is obtained for highly represented variable Ig gene subgroups. Rarely occurring V kappa 4 and V lambda 5 subgroups were not detected. Rearranged Ig variable region genes from each of 19 human B cell lines were also amplified. Comparisons to germline sequences allowed the allocation of rearranged genes to the original Ig genes. This primer set should be very useful for generating large repertoires of rearranged V genes and for amplifying genes of individual B cell clones.
Amino Acid Sequence↗
The sequencing-based typing tool of dbMHC: typing highly polymorphic gene sequences.
The dbMHC resource (http://www.ncbi.nlm.nih.gov/mhc/sbt.cgi?cmd=main) at the National Center for Biotechnology Information (NCBI) has developed an online tool for evaluating the allelic composition of sequencing-based typing (SBT) results of cDNA or genomic sequences. Whether the samples are heterozygous, haploid or a combination of the two, they can be compared with two up-to-date databases of all known alleles of several human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) loci. The results of the submission are returned as a table of potential allele hits, along with the respective base changes and an interactive sequence viewer for close examination of the alignment.
Algorithms↗
Alternative base-calling algorithm for DNA sequencing based on four-label multicolor detection.
A simple base-calling scheme based on four-label multicolor detection is suggested for DNA sequencing. The entire spectra of the dye labels were used for identification. Specifically, the maxima of the emission spectra rather than the intensity ratios at selected wavelengths are used to provide excellent discrimination. Capillary gel electrophoresis was used for the separation of DNA fragments. Data acquisition and analysis compatible with fast and high-throughput imaging detection was accomplished. The accuracy of base calling of PGEM/U DNA from the raw data obtained with 5 nm and 7 nm spectroscopic resolution were 98.4% for 386 bases and 98.4% for 385 bases. Base calling of M13mp18 DNA showed 98.3% accuracy for 420 bases.
Algorithms↗
Comparing protein sequence-based and predicted secondary structure-based methods for identification of remote homologs.
We have compared a novel sequence-structure matching technique, FORESST, for detecting remote homologs to three existing sequence based methods, including local amino acid sequence similarity by BLASTP, hidden Markov models (HMMs) of sequences of protein families using SAM, HMMs based on sequence motifs identified using meta-MEME. FORESST compares predicted secondary structures to a library of structural families of proteins, using HMMs. Altogether 45 proteins from nine structural families in the database CATH were used in a cross-validated test of the fold assignment accuracy of each method. Local sequence similarity of a query sequence to a protein family is measured by the highest segment pair (HSP) score. Each of the HMM-based approaches (FORESST, MEME, amino acid sequence-based HMM) yielded log-odds score for the query sequence. In order to make a fair comparison among these methods, the scores for each method were converted to Z-scores in a uniform way by comparing the raw scores of a query protein with the corresponding scores for a set of unrelated proteins. Z-Scores were analyzed as a function of the maximum pairwise sequence identity (MPSID) of the query sequence to sequences used in training the model. For MPSID above 20%, the Z-scores increase linearly with MPSID for the sequence-based methods but remain roughly constant for FORESST. Below 15%, average Z-scores are close to zero for the sequence-based methods, whereas the FORESST method yielded average Z-scores of 1.8 and 1.1, using observed and predicted secondary structures, respectively. This demonstrates the advantage of the sequence-structure method for detecting remote homologs.
Algorithms↗
Identification to the species level and differentiation between strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR.
A commercially available repetitive-sequence-based PCR (rep-PCR) DNA fingerprinting assay adapted to an automated format, the DiversiLab system, enables rapid microbial identification and strain typing. We explored the performance of the DiversiLab system as a molecular typing tool for 69 Aspergillus isolates (38 A. fumigatus, 15 A. flavus, and 16 A. terreus isolates) had been previously characterized by morphological analysis. Initially, 27 Aspergillus isolates (10 A. fumigatus, 9 A. flavus, and 8 A. terreus isolates) were used as controls to create a rep-PCR-based DNA fingerprint library with the DiversiLab software. Then, 42 blinded Aspergillus isolates were typed using the system. The rep-PCR-based profile revealed 98% concordance with morphology-based identification. rep-PCR-based DNA fingerprints were reproducible and were consistent for DNA from both hyphae and conidia. DiversiLab dendrogram reports correctly identified all A. fumigatus (n = 28), A. terreus (n = 8), and A. flavus (n = 6) isolates in the 42 blinded Aspergillus isolates. rep-PCR-based identification of all isolates was 100% in agreement with the contiguous internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) sequence-based identification of the respective isolates. Additionally, the DiversiLab system could demonstrate strain-level differentiation of A. flavus and A. terreus. Automated rep-PCR may be a time-efficient, effective, easy-to-use, novel genotyping tool for identifying and determining the strain relatedness of fungi. This system may be useful for epidemiological studies, molecular typing, and surveillance of Aspergillus species.
Aspergillus↗