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In a paternity test with 21 short tandem repeats (STRs) an isolated exclusion for the TH01 locus was observed. The probability of paternity or maternity in this case turned out greater than 99,999%. The analysis of sequence indicates only the deletion character of the observed mutation event without any changes in the conservative sequence.
MOTIVATION: Nanopore sequencing by Oxford Nanopore Technologies (ONT) enables direct analysis of DNA and RNA by capturing raw electrical signals. Different nanopore chemistries have varied k-mer lengths, current levels, and standard deviations, which are stored in "k-mer models." In cases where official models are lacking or unsuitable for specific sequencing conditions, tailored k-mer models are crucial to ensure precise signal-to-sequence alignment, analysis and interpretation. The process of transforming raw signal data into nucleotide sequences, known as basecalling, is a fundamental step in nanopore sequencing. RESULTS: In this study, we leverage the move table produced by ONT's basecalling software to create a lightweight de novo k-mer model for RNA004 chemistry. We demonstrate the validity of our custom k-mer model by using it to guide signal-to-sequence alignment analysis, achieving high alignment rates (97.48%) compared to larger default models. Additionally, our 5-mer model exhibits similar performance as the default 9-mer models another analysis, such as detection of m6A RNA modifications. We provide our method, termed Poregen, as a generalizable approach for creation of custom, de novo k-mer models for nanopore signal data analysis. AVAILABILITY AND IMPLEMENTATION: Poregen is an open source package under an MIT license: https://github.com/hiruna72/poregen.
Broad range amplification and sequence analysis of the 16S ribosomal RNA gene was used to identify three spiral-form organisms. The agents were identified as Campylobacter fetus, "Flexispira rappini", and Borrelia burgdorferi, respectively, using either proprietary or public sequence databases. In each case, the rDNA sequence showed 99-100% homology with known sequence data. Sequence-based analysis for each isolate required only 2-3 days, whereas traditional means of identification took 8-12 days to complete. The identification of spirochetes and vibrio-like agents from human clinical samples is often time consuming and results may be difficult to interpret, sometimes due to atypical phenotypic characteristics. Analysis of 16S rDNA or other molecular targets may provide a way to accurately and rapidly characterize isolates that are recalcitrant to speciation.
Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H). Phylogenetic analysis of known sequences of F3'H and F3'5'H indicated that F3'5'H was recruited from F3'H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3'Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3'H activity but surprisingly, three recombinant proteins showed F3'5'H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3'5'H. Furthermore, sequence analysis of these unusual F3'5'H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3'5'H is necessary for the synthesis of 3',4',5'-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3'5'H probably reflects the adaptive value of efficient attraction of insect pollinators.
We used ITS-RFLP and sequence analysis to determine the identities of the fungal endophytes of six terrestrial orchid species from southeastern Queensland, a region previously unexplored in this context. Pure cultures of orchid--colonising fungi were obtained and fungal identities were assessed by means of ITS-PCR, RFLP analysis, sequence comparison, and protocorm colonisation tests. ITS-PCR and RFLP analysis resulted in five main groupings. Sequencing and GenBank comparison of these five groups showed that the fungal endophytes isolated from the three Pterostylis species were probably Thanatephorus species. There was close sequence identity (90%) of the fungus isolated from Acianthus spp. to Epulorhiza repens, suggesting these may be the same fungal species. However, that only E. repens succeeded in colonising protocorms of Thelymitra pauciflora suggests these may be different species of Epulorhiza. Analysis of the ITS and LSU sequences of the fungus isolated from Caladenia carnea showed high identities with a sequence from a Sebacina vermifera originally isolated from Caladenia dilatata. These results show that there is specificity for fungal partners within the orchid genera Acianthus, Caladenia and Pterostylis.
The nucleotide sequences of the 16S rRNA gene (rDNA) in 38 taxa of the genus Staphylococcus were compared phylogenetically. Based on phylogenetic tree analysis, staphylococcal species were divided into 12 cluster groups. These cluster groups were in very good agreement with species groups determined by DNA-DNA reassociation studies. These genealogical classifications were consistent with the results of the production of coagulase or oxidase and with resistance to novobiocin. These suggest that the phylogenetic relationship of the genus Staphylococcus is accurately represented by the results obtained from the sequence analysis of 16S rDNA.
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Carbonyl reductase (EC 1.1.1.184) is one of several monomeric, NADPH-dependent oxidoreductases having wide specificity for carbonyl compounds that are generally referred to as the aldoketoreductases. The grouping of the enzyme into the family has been proposed on the basis of functional similarities and in the absence of structural data. Here, we describe the isolation and characterization of a cDNA clone complementary to human carbonyl reductase mRNA from a human placenta cDNA library constructed in phage lambda gt11. The cDNA consists of 1199 base pairs and contains an open reading frame encoding a protein comprised of 277 amino acids with a Mr of 30,375. The predicted amino acid sequence was confirmed by partial sequence analysis of the carbonyl reductase protein. Comparison of the protein sequence with the primary structures of other aldoketoreductases revealed no significant homologies. A possible homology, on the other hand, exists between carbonyl reductase and "short" subunit alcohol/polyol dehydrogenases.
The aim of this study was to analyse with sequence analysis physician-patient dialogue surrounding patients' expression of emotional cues. Two samples, sample 1 consisting of 36 cancer patient consultations conducted by four oncologists, and sample 2 consisting 79 consultations of haematology patients conducted by nine specialists, were audiotaped and coded with the Roter Interaction Analysis System (RIAS). Sequence analysis by means of a generalized sequential querier (GSEQ) was applied to the coded data. Lag sequential analysis (analysed using RIAS categories) showed that certain behaviours of physicians corresponded with patients' expressions of concern. Physicians in both samples used silence and minimal encouragers before patient concern. The oncologists also used optimistic and affirming responses. The most common physician responses to patients' concern were minimal encouragers or affirming and optimistic responses. Sequence analysis based on RIAS coding appears to be a promising method for the study of doctor-patient dialogue and should be utilized more in studies of the communication process in medical consultations.
Comparative sequence analysis of IS50 transposase-related protein sequences in conjunction with known structural, biochemical, and genetic data was used to determine domains and residues that play key roles in IS50 transposase function. BLAST and ClustalW analyses have been used to find and analyze six complete protein sequences that are related to the IS50 transposase. The protein sequence identity of these six homologs ranged from 25 to 55% in comparison to the IS50 transposase. Homologous motifs were found associated with each of the three catalytic residues. Residues that play roles in transposase-DNA binding, protein autoregulation, and DNA hairpin formation were also found to be conserved in addition to other residues of unknown function. On the other hand, some homologous sequences did not appear to be competent to encode the inhibitor regulatory protein. The results were also used to compare the IS50 transposase with the more distantly related transposase encoded by IS10.
The existence of Helicobacter pylori in the biliary tract was investigated. Seven bile samples were included in this study. Among them, six bile samples were collected by percutaneous transhepatic cholangiodrainage and the other by needle aspiration during cholecystectomy. Using nested PCR with two sets of primers homologous to the urease A gene, Helicobacter pylori DNA was detected. Three samples, one from a patient with advanced gastric cancer involving the pancreatic head and two from patients with pancreatic head tumor, were found to be positive for Helicobacter pylori DNA. On the other hand, three samples from patients with cholangiocarcinoma and one from a patient with chronic cholecystitis were all negative. To further verify the specificity of our PCR analysis, partial sequences of the PCR products from the three positive samples were analyzed by direct sequencing. Several silent mutations and a missense mutation (AAA to AGA; Lys-164 to Arg-164) were identified in the urease A gene. We conclude that Helicobacter pylori DNA can be easily detected in the bile samples. The possibility of asymptomatic cholangitis caused by this organism requires further investigation.
The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3' end and 5' end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month.
Multiple brain tumors of diffuse large B cell lymphoma (DLBL) were observed in a 75-year-old man with Waldenström's macroglobulinemia (WM). Paravertebral and multiple subcutaneous nodules occurred in succession and he died 4 months after onset. We investigated B cell monoclonality by means of polymerase chain reaction (PCR) analysis and sequencing of the immunoglobulin heavy chain of paraffin-embedded sections. The PCR product of the brain tumors showed a different rearrangement pattern from those of the other sections. The co-occurrence of DLBL with WM is rare, and some investigators have examined the clonality of the two malignancies. This case is important because DLBL brain tumors co-occurred with WM, enabling us to prove that DLBL and WM have different clonality.
Escherichia coli HX88108, which is resistant to cefoperazone(CPZ), was isolated from a severely infected patient. We studied genetical basis of beta-lactamase produced in E. coli HX88108 by pFL25, one of the recombinant plasmid of pFC. Largescale pFL25 plasmid was extracted, purified, and cleaved with restriction endonuclease EcoR I, Sal I, Pvu I, then subcloned into vector pUC19 as 1.9 kb, 0.9 kb, 0.65 kb fragments respectively. Recomminant plasmids were selected by alpha-complementation and determined by restriction endonuclease analysis. DNA sequencing was performed by the dideoxy polymerase chain termination method. Partial nucleotide sequence(1-78 nucleotide position) of the gene was found to be highly homologous (97%) with the gene coding for TEM-52 extended spectrum beta-lactamase of K. pneumonise, suggesting that the beta-lactamase coded by the cefoperazone resistant gene might be derived from TEM-type beta-lactamase.
VISA (VIsual Sequence Analysis) is a software package that displays global similarities within a set of related protein sequences. The program identifies amino acid patterns that are common to many members of the set of sequences and displays them as a series of histograms. Individual peaks on the display can be assigned a color and analogous peaks in the other sequences are then automatically marked in the same color. This can be repeated for each significant peak and leads to a display in which major matching segments of multiple amino acid sequences appear as dominant peaks of the histograms with matching colors. These peaks usually correspond to the conserved sequence motifs that are characteristic of particular proteins. An extensive set of software tools is included to help the localization, visualization and analysis of the global similarities displayed. VISA provides a graphic overview of the sequence similarity that can help to understand the architecture of the protein family and can be helpful while designing experiments to probe function.
The 16S rRNA gene sequences of the type strains of Aeromonas enteropelogenes and Aeromonas ichthiosmia were determined by polymerase chain reaction direct sequencing in order to clarify their interrelationships with other aeromonad species. On the basis of 16S rRNA gene sequence analysis, A. enteropelogenes and A. ichthiosmia were found to be identical to Aeromonas trota and Aeromonas veronii, respectively.
We report the use of chemical derivatization with MALDI-MS/MS analysis for de novo sequence analysis. Using three frequently used homology-based search algorithms, we were able to identify more than 40 proteins from banana, a nonmodel plant with unsequenced genome. Furthermore, this approach allowed the identification of different isoforms. We also observed that the identification score obtained varied according to the position of the peptide sequences in the query using the MS-Blast algorithm.
The packing orientations of the 8 transmembrane (TM) segments that line the central, aqueous transport channel within tetracycline resistance proteins (TetA) have been established. However, the orientations of the remaining 4 segments, TMs 3, 6, 9, and 12, located at the periphery, and away from the transport channel, have not yet been determined. In this study, the packing orientation of TM6 within the class C TetA protein encoded by plasmid pBR322 was evaluated by substitution mutagenesis and analysis of sequence conservation and amphipathicity. The combined data support a model in which the conserved and polar face of the TM6 alpha-helix containing Asn170 and Asn173 orients towards channel-lining TM segments, and the relatively non-conserved and hydrophobic face of TM6 points towards membrane lipids.