Generation Of Consensus Sequences Research Articles

Abstract 437: Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel

Abstract Background: The rapid spread of COVID-19 has resulted in an urgent need for effective diagnostic and therapeutic strategies against SARS-CoV-2. Next-generation sequencing (NGS) is a powerful tool in the identification and characterization of this pathogen and genomic information may aid in understanding the mechanisms of therapeutic resistance, vaccine escape, virulence, and pathogenicity. The Ion AmpliSeq SARS-CoV-2 Research Panel is a targeted NGS solution that facilitates sequence analysis of the SARS-CoV-2 genome. Paired with a bioinformatics assembly and variant calling pipelines, this assay allows for accurate characterization of the dominant SARS-CoV-2 variant. This assay’s performance was analytically validated for the detection of mutations (substitutions, insertions, and deletions) in RNA derived from nasopharyngeal (NP) swabs. Method: The Ion AmpliSeq SARS-CoV-2 Research panel consists of two primer pair pools generating 237 amplicons specific to the SARS-CoV-2 virus. Reverse transcription of the RNA was performed using the SuperScript VILO cDNA Synthesis kit. Library preparation was then completed using the Ion AmpliSeq Library Kit Plus kit. The final library was quantified, normalized, pooled, and sequenced. Raw sequencing data was aligned to the AmpliSeq SARS-CoV-2 Research panel, using the MN908947.3 reference genome. Variants were called using the Torrent Variant Caller and annotated using the COVID19AnnotateSnpEff plugin. The reference-guided iterative assembler IRMA was used to produce a single consensus sequence consisting of the reference genome sequence modified to include sequence variations supported by the reads. The Pangolin COVID-19 lineage assigner software tool was used to assign SARS-CoV-2 lineage. Analytical validation was completed using controls (Twist Biosciences, BEI Resources, ATCC) and RNA derived from NP swabs. Accuracy and specificity were examined by evaluating the correctness of calling true negative variants compared to false positive and all other variant calls, respectively. Precision and limit of detection (LoD) were examined by evaluating the concordance of variants across replicate samples. Limit of Blank (LoB) was calculated as the 95th percentile of reads per amplicon in the negative samples. Results: Accuracy of base calling, specificity, and precision were 100% for SNVs, insertions, and deletions above 25% allele frequency. LoD was determined to be 576 viral copies/mL. LoB was determined to be 202 reads per amplicon. Pangolin lineage assignment was 100% for all samples. Conclusions: This panel accurately characterizes SARS-CoV-2 variants, allowing for accurate consensus sequence generation, mutation annotation, and lineage assignment. Citation Format: Desiree Unselt, Katherine Knudsen, Christopher Rounds, Janet Doolittle-Hall, Fernando Torres, Jennifer Sims, Jennifer Mason. Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 437.

SnackNTM: An Open-Source Software for Sanger Sequencing-based Identification of Nontuberculous Mycobacterial Species.

BackgroundSequence-based identification is one of the most effective methods for species-level identification of nontuberculous mycobacteria (NTM). However, it is time-consuming because of the bioinformatics processes involved, including sequence trimming, consensus sequence generation, and public database searches. We developed a simple and fully automated software that enabled species-level identification of NTM from trace files, SnackNTM (https//github.com/Young-gonKim/SnackNTM).MethodsJAVA programing language was used for software development. The SnackNTM diagnostic algorithm utilized 16S rRNA gene sequences, according to the Clinical & Laboratory Standards Institute guidelines, and an rpoB gene region was adjunctively utilized to narrow down the species. The software performance was validated using trace files of 234 clinical cases, comprising 217 consecutive cases and 17 additionally selected cases of unique species.ResultsSnackNTM could analyze multiple cases at once, and all the bioinformatics processes required for sequence-based NTM identification were automatically performed with a single mouse click. SnackNTM successfully identified 95.9% (208/217) of consecutive clinical cases, and the results showed 99.0% (206/208) agreement with manual classification results. SnackNTM successfully identified all 17 cases of unique species. In a processing time comparison test, the analysis and reporting of 30 cases, which took 150 minutes manually, took only 40 minutes with SnackNTM.ConclusionsSnackNTM is expected to reduce the workload for NTM identification, especially in clinical laboratories that process large numbers of cases.

A Guide to Using FASTPCR Software for PCR, In Silico PCR, and Oligonucleotide Analysis.

The FastPCR software is an integrated tool environment for PCR primer and probe design and for prediction of oligonucleotide properties. The software provides comprehensive tools for designing primers for most PCR and perspective applications, including standard, multiplex, long-distance, inverse, real-time with TaqMan probe, Xtreme Chain Reaction (XCR), group-specific, overlap extension PCR for multifragment assembling cloning, and isothermal amplification (Loop-mediated Isothermal Amplification). A program is available to design specific oligonucleotide sets for long sequence assembly by ligase chain reaction and to design multiplexed of overlapping and nonoverlapping DNA amplicons that tile across a region(s) of interest for targeted next-generation sequencing, competitive allele-specific PCR (KASP)-based genotyping assay for single-nucleotide polymorphisms and insertions and deletions at specific loci, among other features. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. FastPCR includes various bioinformatics tools for analysis and searching of sequences, restriction I-II-III-type enzyme endonuclease analysis, and pattern searching. The program also supports the assembly of a set of contiguous sequences, consensus sequence generation, and sequence similarity and conservancy analysis. FastPCR performs efficient and complete detection of various repeat types with visual display. FastPCR allows for sequence file batch processing that is essential for automation. The software is available for download at https://primerdigital.com/fastpcr.html and online version at https://primerdigital.com/tools/pcr.html .

Clinical Significance of Clonal Hematopoiesis in the Setting of Autologous Stem Cell Transplantation for Lymphoma

Clinical Significance of Clonal Hematopoiesis in the Setting of Autologous Stem Cell Transplantation for Lymphoma

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field.

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

In silico analysis of four structural proteins of aphthovirus serotypes revealed significant B and T cell epitopes

Foot and Mouth disease (FMD) is economically devastating, highly contagious transboundry viral disease of livestock with 100% morbidity, rapid spread and severe production losses in animals. The FMDV has seven different serotypes. There is no vaccine that can protect animals from all serotypes. Hence, it is need of the day to develop a vaccine that protects animals from hetrologous challenge. In this study, we used immunoinformatics approach to find T and B-cell epitopes that will help to construct a universal vaccine for FMDV. For this purpose, first we constructed a consensus sequence for four structural proteins (VP1, VP2, VP3 and VP4) of aphthovirus for seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3). Various computational tools were used to perform multiple sequence alignment to identify the conserved regions, generation of consensus sequence through conserved regions, structures prediction and finally prediction of B and T cell epitopes. We predicted 5 B cell and 18 T cell epitopes. Finally a GPGPG spacer was used to join these epitopes to decrease binding affinity around the core binding regions. Hence, our study identified the epitopes which can be used to develop cross protective vaccines against all the fatal strains of Aphthovirus which can easily protect all the serotypes. Though, successful In vivo and In vitro studies are required to determine the genuine strength of our predicted epitopes against the fatal strains of Aphthovirus.

Identification of Tibetan medicine "Dida" of Gentianaceae using DNA barcoding

The ITS2 barcode was used toidentify Tibetan medicine "Dida", and tosecure its quality and safety in medication. A total of 13 species, 151 experimental samples for the study from the Tibetan Plateau, including Gentianaceae Swertia, Halenia, Gentianopsis, Comastoma, Lomatogonium ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V3.7.1. The Kimura 2-Parameter (K2P) distances were calculated using MEGA 6.0. The neighbor-joining (NJ) phylogenetic trees were constructed. There are 31 haplotypes among 231 bp after alignment of all ITS2 sequence haplotypes, and the average G±C content of 61.40%. The NJ tree strongly supported that every species clustered into their own clade and high identification success rate, except that Swertia bifolia and Swertia wolfangiana could not be distinguished from each other based on the sequence divergences. DNA barcoding could be used as a fast and accurate identification method to distinguish Tibetan medicine "Dida" to ensure its safe use.

Molecular identification of Uncaria (Gouteng) through DNA barcoding.

BackgroundWhile DNA barcoding is an important technology for the authentication of the botanical origins of Chinese medicines, the suitable markers for DNA barcoding of the genus Uncaria have not been reported yet. This study aims to determine suitable markers for DNA barcoding of the genus Uncaria (Gouteng).MethodsGenomic DNA was extracted from the freshly dried leaves of Uncaria plants by a Bioteke’s Plant Genomic DNA Extraction Kit. Five candidate DNA barcode sites (ITS2, rbcL, psbA–trnH, ITS, and matK) were amplified by PCR with established primers. The purified PCR products were bidirectionally sequenced with appropriate amplification primers in an ABI-PRISM3730 instrument. The candidate DNA barcodes of 257 accessions of Uncaria in GenBank were aligned by ClustalW. Sequence assembly and consensus sequence generation were performed with CodonCode Aligner 3.7.1. The identification efficiency of the candidate DNA barcodes was evaluated with BLAST and nearest distance methods. The interspecific divergence and intraspecific variation were assessed by the Kimura 2-Parameter model. Genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0.ResultsThe accessions of the five candidate DNA barcodes from 11 of 12 species of Uncaria in China and four species from other countries were included in the analysis, while 54 of total accessions were submitted to GenBank. In a comparison of the interspecific genetic distances of the five candidate barcodes, psbA–trnH exhibited the highest interspecific divergence based on interspecific distance, theta prime, and minimum interspecific distance, followed by ITS2. The distribution of the interspecific distance of ITS2 and psbA–trnH was higher than the corresponding intraspecific distance. Additionally, psbA–trnH showed 95.9 % identification efficiency by both the BLAST and nearest distance methods regardless of species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method.ConclusionWhile psbA–trnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbA–trnH was a more suitable barcode for authentication of Uncaria macrophylla.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-015-0072-7) contains supplementary material, which is available to authorized users.

Generation of high confidence HLA genotyping and consensus sequences for class I HLA loci using the NGS-based Omixon Holotype HLA typing system and the Illumina Miseq platform

Aim Generate HLA genotyping and high confidence consensus sequences for 50 samples at HLA-A, B and C loci using the Omixon Holotype HLA typing system. Methods The Omixon Holotype HLA typing kit was used to generate sequencing libraries for 50 samples for HLA-A, B and C loci, and sequenced on an Illumina MiSeq. HLA typing and consensus sequence generation was performed using Omixon Twin, GenDx NGSengine and a custom pipeline, which relies on stringently mapping reads to the genotyped alleles and using SAMtools to generate the consensus. Consensus sequences generated by each method were aligned pairwise to determine concordance using a semi-global Needleman–Wunsch algorithm. When the sequences generated by each method were concordant, sequences were aligned to the IMGT reference sequence of the genotyped allele, to annotate novel alleles and annotate the sequence of incomplete alleles. When sequences generated by each method were discordant, sequences were aligned to the genotyped allele for manual inspection and assessment of algorithm performance. Results HLA genotyping results for all 300 alleles (50 samples at 3 loci) were 100% concordant between NGSengine and Omixon Twin, after manually resolving discrepancies (6/300). Consensus sequences were generated for every allele (300) encompassing 31 unique HLA-A, 52 unique HLA-B and 31 unique HLA-C alleles. Our analysis facilitated the annotation of full gene sequences for 11 HLA alleles for which there is no complete IMGT reference sequence (A∗02:04, B∗14:03, B∗15:08:01, B∗15:30, B∗27:02:01, B∗35:04:01, B∗35:43:01, B∗39:09:01, B∗51:08:01, B∗58:02 and C∗07:18) and facilitated the discovery of two novel HLA-A alleles, with intronic substitutions. For the remaining alleles, consensus sequences were compared against the reference sequences of the genotyped alleles and occasional differences between consensus sequences and reference sequences were manually inspected to evaluate algorithm performance. It has been determined that depth of coverage is critical in phasing and base calling, which is necessary to generate credible consensus sequences. Conclusions Generation of high confidence HLA class I consensus sequences is possible using the Omixon Holotype HLA typing system provided that adequate depth of uniform coverage is obtained from sequencing. D. Monos: Other (Identify); Company/Organization; Omixon Royalties.

FASconCAT-G: extensive functions for multiple sequence alignment preparations concerning phylogenetic studies.

BackgroundPhylogenetic and population genetic studies often deal with multiple sequence alignments that require manipulation or processing steps such as sequence concatenation, sequence renaming, sequence translation or consensus sequence generation. In recent years phylogenetic data sets have expanded from single genes to genome wide markers comprising hundreds to thousands of loci. Processing of these large phylogenomic data sets is impracticable without using automated process pipelines. Currently no stand-alone or pipeline compatible program exists that offers a broad range of manipulation and processing steps for multiple sequence alignments in a single process run.ResultsHere we present FASconCAT-G, a system independent editor, which offers various processing options for multiple sequence alignments. The software provides a wide range of possibilities to edit and concatenate multiple nucleotide, amino acid, and structure sequence alignment files for phylogenetic and population genetic purposes. The main options include sequence renaming, file format conversion, sequence translation between nucleotide and amino acid states, consensus generation of specific sequence blocks, sequence concatenation, model selection of amino acid replacement with ProtTest, two types of RY coding as well as site exclusions and extraction of parsimony informative sites. Convieniently, most options can be invoked in combination and performed during a single process run. Additionally, FASconCAT-G prints useful information regarding alignment characteristics and editing processes such as base compositions of single in- and outfiles, sequence areas in a concatenated supermatrix, as well as paired stem and loop regions in secondary structure sequence strings.ConclusionsFASconCAT-G is a command-line driven Perl program that delivers computationally fast and user-friendly processing of multiple sequence alignments for phylogenetic and population genetic applications and is well suited for incorporation into analysis pipelines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-014-0081-x) contains supplementary material, which is available to authorized users.

Identification of Placenta hominis and its adulterants using COI barcode

In order to provide a new method for the identification of Placenta hominis, the COI barcode has been employed to identify the P. hominis medicinal materials and its adulterants. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. NJ tree was constructed by MEGA6.0 software. COI sequences can be successfully obtained from all experimental samples. The intra-specific variation and inter-specific divergence were calculated. The average intra-specific K2P distance of P. hominis was 0.001 and the maximum intra-specific distance was 0.008. The cluster dendrogram constructed can be seen that the same genus is together, and distinguished from its adulterants. It is concluded that P. hominis and its adulterants can be correctly identified by DNA barcoding method.

Identification of Cimicifugae Rhizoma and its adulterants using ITS2 sequence

In order to identify Cimicifugae Rhizoma from its adulterants and to ensure its safe use, the internal transcribed spacer 2 (ITS2) sequence of Cimicifugae Rhizoma and its adulterants were amplified and bidirectionally sequenced by DNA barcoding technology. Sequence assembly and consensus sequence generation were performed by the CodonCode Aligner V3.7.1. The genetic distances were computed by MEGA 5.0. Identification analyses were performed using neighbor-joining (NJ) methods. The length of ITS2 sequence of the three origin plants of Cimicifugae Rhizoma include Cimicifuga heracleifolia, C. foetida, C. dahurica was 217, 219 and 219 bp, respectively. Their intraspecific genetic distance was much lower than the interspecific genetic distance with their closely related species. The NJ tree of ITS2 indicated that the three origin plants of Cimicifugae Rhizoma formed a monophyletic clade, Cimicifugae Rhizoma and its adulterants could be distinguished clearly. The authors proposed that ITS2 sequence was suitable for the authentication of Cimicifugae Rhizoma and its adulterants.

Identification of Schisandra chinensis with white fruits based on ITS2 sequences

To analyse a special kind of Schisandra chinensis with the white fruit using ITS2 barcode at molecular levels. ITS2 regions were sequenced bidirectionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner, MEGA 5.0 software was used to align the sequences. The ITS2 secondary structure was predicted using ITS2 web server, BLAST 1 method was used to identify the S. chinensis with the white fruit. The length of the ITS2 sequence was 231 bp. And the sample was identified as S. chinensis using the method of BLAST 1. Their mean interspecific genetic distance (K2P distance) among the populations of the S. chinensis with the white fruit and S. chinensis was far lower than the mean interspecific genetic distance between the S. chinensis and S. sphenanthera. By using ITS2 the S. chinensis with the white fruit was identified as S. chinensis, and the ITS2 barcode could be used to identify S. chinensis and S. sphenanthera.

Molecular identification of Cynomorii Herba using ITS2 DNA barcoding

To identify the Cynomorii Herba and its analogues species using DNA barcoding technique. Total genomic DNA extracted from all materials using the DNA extraction kit. The internal transcribed spacer 2 (ITS2) regions were amplified using polymerase chain reaction (PCR), and purified PCR products were sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner 3.7.1. The Kimura 2-Parameter (K2P) distances and GC content were computed using MEGA 5. 0. Species identification analyses were conducted through the species identification system for traditional Chinese medicine and neighbor-joining (NJ) trees. The ITS2 sequence lengths of Cynomorii Herba were 229 bp. The average intra-specific genetic distances of Cynomorii Herba were 0.003. The average inter-specific genetic distances between Cynomorii Herba and its adulterants species were 0.760. The results showed that the minimum inter-specific divergence is larger than the maximum intra-specific divergence. The species identification system for traditional Chinese medicine and NJ trees results indicated that Cynomorii Herba and its adulterants species can be easily identification. The ITS2 region is an efficient barcode for identification of Cynomorii Herba, which provide a new technique to ensure clinical safety in utilization of traditional Chinese medicine.

Identification of Junci Medulla using DNA barcoding technique

To identify Junci Medulla using the ITS2 barcode. The ITS2 regions of Juncus effuses and its closely related species were PCR amplified and sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance, PWG Distance and neighbor-joining (NJ) methods. The intra-specific genetic distances of J. effuses were ranged from 0 to 0.005, which were far lower than inter-specific genetic distances between J. effuses and its closely related species (0.215-0.614). All the four methods showed that ITS2 could discriminate J. effuses from its closely related species correctly. The ITS2 region is an efficient barcode for authentication of Junci Medulla, and our study further confirmed the ability of ITS2 to identify traditional Chinese medicinal materials.

Using ITS2 barcode to identify Ephedrae Herba

To identify Ephedrae Herba using the ITS2 barcode and to secure its quality and safety in medication. Total genomic DNA was isolated from Ephedrae Herba and its closely related species. Nuclear DNA ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance and neighbor-joining (NJ) methods. The intra-specific genetic distances of Ephedrae Herba were ranged from 0 to 0.002. The inter-specific genetic distances between Ephedrae Herba and its closely related species were ranged from 0.004 to 0.034. All the three methods showed that ITS2 could discriminate Ephedrae Herba from its closely related species correctly. The ITS2 region is suitable to be used for authentication of Ephedrae Herba, and our study further confirmed the effectiveness of ITS2 to identify traditional Chinese medicinal materials.

Applying DNA barcoding technique to identify Menthae Haplocalycis Herba

To identify Menthae Haplocalycis Herba and its closely related species using DNA barcoding technique. Total genomic DNA was isolated from Mentha canadensis and its closely related species. Nuclear DNA ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V3.0. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance and neighbor-joining (NJ) methods. The intra-specific genetic distances of M. canadensis were ranged from 0 to 0.006, which were lower than inter-specific genetic distances between M. canadensis and its closely related species (0.071-0.231). All the three methods showed that ITS2 could discriminate M. canadensis from its closely related species correctly. The ITS2 region is an efficient barcode for identification of Menthae Haplocalycis Herba, which provides a scientific basis for fast and accurate identification of the herb.

ANDES: Statistical tools for the ANalyses of DEep Sequencing.

BackgroundThe advancements in DNA sequencing technologies have allowed researchers to progress from the analyses of a single organism towards the deep sequencing of a sample of organisms. With sufficient sequencing depth, it is now possible to detect subtle variations between members of the same species, or between mixed species with shared biomarkers, such as the 16S rRNA gene. However, traditional sequencing analyses of samples from largely homogeneous populations are often still based on multiple sequence alignments (MSA), where each sequence is placed along a separate row and similarities between aligned bases can be followed down each column. While this visual format is intuitive for a small set of aligned sequences, the representation quickly becomes cumbersome as sequencing depths cover loci hundreds or thousands of reads deep.FindingsWe have developed ANDES, a software library and a suite of applications, written in Perl and R, for the statistical ANalyses of DEep Sequencing. The fundamental data structure underlying ANDES is the position profile, which contains the nucleotide distributions for each genomic position resultant from a multiple sequence alignment (MSA). Tools include the root mean square deviation (RMSD) plot, which allows for the visual comparison of multiple samples on a position-by-position basis, and the computation of base conversion frequencies (transition/transversion rates), variation (Shannon entropy), inter-sample clustering and visualization (dendrogram and multidimensional scaling (MDS) plot), threshold-driven consensus sequence generation and polymorphism detection, and the estimation of empirically determined sequencing quality values.ConclusionsAs new sequencing technologies evolve, deep sequencing will become increasingly cost-efficient and the inter and intra-sample comparisons of largely homogeneous sequences will become more common. We have provided a software package and demonstrated its application on various empirically-derived datasets. Investigators may download the software from Sourceforge at https://sourceforge.net/projects/andestools.

The TIGR Plant Transcript Assemblies database

The TIGR Plant Transcript Assemblies (TA) database () uses expressed sequences collected from the NCBI GenBank Nucleotide database for the construction of transcript assemblies. The sequences collected include expressed sequence tags (ESTs) and full-length and partial cDNAs, but exclude computationally predicted gene sequences. The TA database includes all plant species for which more than 1000 EST or cDNA sequences are publicly available. The EST and cDNA sequences are first clustered based on an all-versus-all pairwise sequence comparison, followed by the generation of consensus sequences (TAs) from individual clusters. The clustering and assembly procedures use the TGICL tool, Megablast and the CAP3 assembler. The UniProt Reference Clusters (UniRef100) protein database is used as the reference database for the functional annotation of the assemblies. The transcription orientation of each TA is determined based on the orientation of the alignment with the best protein hit. The TA sequences and annotation are available via web interfaces and FTP downloads. Assemblies can be retrieved by a text-based keyword search or a sequence-based BLAST search. The current version of the TA database is Release 2 (July 17, 2006) and includes a total of 215 plant species.

Delineation of Type I Protein Kinase A-selective Signaling Events Using an RI Anchoring Disruptor

Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14-18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.