Microcomputer programs for DNA sequence analysis.

Computer programs are described which allow (a) analysis of DNA sequences to be performed on a laboratory microcomputer or (b) transfer of DNA sequences between a laboratory microcomputer and another computer system, such as a DNA library. The sequence analysis programs are interactive, do not require prior experience with computers and in many other respects resemble programs which have been written for larger computer systems (1-7). The user enters sequence data into a text file, accesses this file with the programs, and is then able to (a) search for restriction enzyme sites or other specified sequences, (b) translate in one or more reading frames in one or both directions in order to find open reading frames, or (c) determine codon usage in the sequence in one or more given reading frames. The results are given in table format and a restriction map is generated. The modem program permits collection of large amounts of data from a sequence library into a permanent file on the microcomputer disc system, or transfer of laboratory data in the reverse direction to a remote computer system.     

A computer program for comparative analysis of nucleic acid sequences.

The programs offer the possibility of comparing pairs of homologous sequences in order to find out percentage of homology, number of identical and deviating nucleotides, of transitions and transversions and, derived from these, KNUC-values according to Kimura (1) and the corresponding standard error sigmaK. The sequences can be printed in pairs underneath each other, homologies are indicated by asterisks between the identical nucleotides. Out of a set of homologous sequences stored on a disk any number of sequences can be compared in pairs in this way, and a matrix containing either the percentage of homology values, the number of deviating nucleotides or the KNUC-values together with the corresponding standard errors can be sent to screen, printer or disk. A program will be available soon which creates a dendrogram representing the similarity between the sequences by use of an average linkage clustering method deduced from this matrix. The programs are written for Apple II computers using UCSD-PASCAL and for Sirius I/Victor 9000 computers using TURBO-PASCAL.     

利用VBA查找核酸数据库DNA保守序列

采用VBA编写了查找核酸数据库保守序列的四个相关程序,“导入DNA序列”程序可以将Fasta格式的DNA序列文本文件存放到Excel Sheetl的A列中,保留每个序列的Gi号,删除多余的注释部分;“整理DNA序列”程序可以将DNA序列Gi号存放到A列中,B列为对应Gi号的完整序列;“DNA随机序列”程序可以产生DNA随机序列;“发现DNA保守序列”程序可以将随机序列与下载的DNA序列比对,查找每一种随机序列的出现频率.以大豆基因组序列为实例,说明了这些程序的应用方法.该程序弥补了流行序列比对软件的不足,为PCR设计引物、分析基因功能以及种质资源鉴定等方面提供新的工具.     

核酸顺序的计算机辅助分析系统

本文介绍了一个在微机(IBM PC)上实现的、用于核酸顺序分析的计算机程序系统.该系统由三个层次和18个功能块构成,菜单及人机对话使得用户能较快地掌握和使用它.在编程中,采用了树结构、先进后出栈和稀疏矩阵等数据结构技巧,运用了Bayes法等统计分析方法,Kruskal算法和Floyd算法等一系列图论方法也被得到应用,这个软件系统的推出对于分子生物学研究具有一定的积极作用.     

ESPript: analysis of multiple sequence alignments in PostScript.

MOTIVATION: The program ESPript (Easy Sequencing in PostScript) allows the rapid visualization, via PostScript output, of sequences aligned with popular programs such as CLUSTAL-W or GCG PILEUP. It can read secondary structure files (such as that created by the program DSSP) to produce a synthesis of both sequence and structural information. RESULTS: ESPript can be run via a command file or a friendly html-based user interface. The program calculates an homology score by columns of residues and can sort this calculation by groups of sequences. It offers a palette of markers to highlight important regions in the alignment. ESPript can also paste information on residue conservation into coordinate files, for subsequent visualization with a graphics program. AVAILABILITY: ESPript can be accessed on its Web site at http://www.ipbs.fr/ESPript. Sources and helpfiles can be downloaded via anonymous ftp from ftp.ipbs.fr. A tar file is held in the directory pub/ESPript.     

A novel DNAseq program for enhanced analysis of Illumina GAII data: a case study on antibody complementarity-determining regions

High-throughput DNA sequencing technologies are increasingly becoming powerful systems for the comprehensive analysis of variations in whole genomes or various DNA libraries. As they are capable of producing massive collections of short sequences with varying lengths, a major challenge is how to turn these reads into biologically meaningful information. The first stage is to assemble the short reads into longer sequences through an in silico process. However, currently available software/programs allow only the assembly of abundant sequences, which apparently results in the loss of highly variable (or rare) sequences or creates artefact assemblies. In this paper, we describe a novel program (DNAseq) that is capable of assembling highly variable sequences and displaying them directly for phylogenetic analysis. In addition, this program is Microsoft Windows-based and runs by a normal PC with 700MB RAM for a general use. We have applied it to analyse a human naive single-chain antibody (scFv) library, comprehensively revealing the diversity of antibody variable complementarity-determining regions (CDRs) and their families. Although only a scFv library was exemplified here, we envisage that this program could be applicable to other genome libraries.     

Methylation blockage and other improvements to a comprehensive DNA analysis program.

A comprehensive DNA analysis computer program was described in the second special issue of Nucleic Acids Research on the applications of computers to research on nucleic acids by Stone and Potter (1). Criteria used in designing the program were user friendliness, ability to handle large DNA sequences, low storage requirement, migratability to other computers and comprehensive analysis capability. The program has been used extensively in an industrial-research environment. This paper talks about improvements to that program. These improvements include testing for methylation blockage of restriction enzyme recognition sites, homology analysis, RNA folding analysis, integration of a large DNA database (GenBank), a site specific mutagenesis analysis, a protein database and protein searching programs. The original design of the DNA analysis program using a command executive from which any analytical programs can be called, has proven to be extremely versatile in integrating both developed and outside programs to the file management system employed.     

Web-based programs for the display and analysis of transmembrane alpha-helices in aligned protein sequences

We developed novel programs for displaying and analyzing the transmembrane alpha-helical segments (TMSs) in the aligned sequences of homologous integral membrane proteins. TMS_ALIGN predicts the positions of putative TMSs in multiply aligned protein sequences and graphically shows the TMSs in the alignment. TMS_SPLIT (1). predicts the positions of TMSs for each sequence; (2). allows a user to select proteins with a specified number of TMSs, and (3). splits the sequences into groups of TMSs of equal numbers. TMS_CUT works like TMS_SPLIT, but it can cut sequences with any combination of TMSs. The BASS program similarly allows comparison of protein repeat elements, equivalent to TMS_SPLIT plus IC, but it provides the comparison data expressed in BLAST E values. These programs, together with the IntraCompare program, facilitate the identification of repeat sequences in integral membrane proteins. They also facilitate the estimation of protein topology and the determination of evolutionary pathways.     

Prediction of common folding structures of homologous RNAs.

We have developed an algorithm and a computer program for simultaneously folding homologous RNA sequences. Given an alignment of M homologous sequences of length N, the program performs phylogenetic comparative analysis and predicts a common secondary structure conserved in the sequences. When the structure is not uniquely determined, it infers multiple structures which appear most plausible. This method is superior to energy minimization methods in the sense that it is not sensitive to point mutation of a sequence. It is also superior to usual phylogenetic comparative methods in that it does not require manual scrutiny for covariation or secondary structures. The most plausible 1-5 structures are produced in O(MN2 + N3) time and O(N2) space, which are the same requirements as those of widely used dynamic programs based on energy minimization for folding a single sequence. This is the first algorithm probably practical both in terms of time and space for finding secondary structures of homologous RNA sequences. The algorithm has been implemented in C on a Sun SparcStation, and has been verified by testing on tRNAs, 5S rRNAs, 16S rRNAs, TAR RNAs of human immunodeficiency virus type 1 (HIV-1), and RRE RNAs of HIV-1. We have also applied the program to cis-acting packaging sequences of HIV-1, for which no generally accepted structures yet exist, and propose potentially stable structures. Simulation of the program with random sequences with the same base composition and the same degree of similarity as the above sequences shows that structures common to homologous sequences are very unlikely to occur by chance in random sequences.     

Gene recognition by combination of several gene-finding programs

MOTIVATION: A number of programs have been developed to predict the eukaryotic gene structures in DNA sequences. However, gene finding is still a challenging problem. RESULTS: We have explored the effectiveness when the results of several gene-finding programs were re- analyzed and combined. We studied several methods with four programs (FEXH, GeneParser3, GEN-SCAN and GRAIL2). By HIGHEST-policy combination method or BOUNDARY method, approximate correlation (AC) improved by 3- 5% in comparison with the best single gene-finding program. From another viewpoint, OR-based combination of the four programs is the most reliable to know whether a candidate exon overlaps with the real exon or not, although it is less sensitive than GENSCAN for exon-intron boundaries. Our methods can easily be extended to combine other programs. AVAILABILITY: We have developed a server program (Shirokane System) and a client program (GeneScope) to use the methods. GeneScope is available through a WWW site (http://gf.genome.ad.jp/). CONTACT: katsu,takagi@ims.u-tokyo.ac.jp      

Mind the gaps: evidence of bias in estimates of multiple sequence alignments

Multiple sequence alignment (MSA) is a crucial first step in the analysis of genomic and proteomic data. Commonly occurring sequence features, such as deletions and insertions, are known to affect the accuracy of MSA programs, but the extent to which alignment accuracy is affected by the positions of insertions and deletions has not been examined independently of other sources of sequence variation. We assessed the performance of 6 popular MSA programs (ClustalW, DIALIGN-T, MAFFT, MUSCLE, PROBCONS, and T-COFFEE) and one experimental program, PRANK, on amino acid sequences that differed only by short regions of deleted residues. The analysis showed that the absence of residues often led to an incorrect placement of gaps in the alignments, even though the sequences were otherwise identical. In data sets containing sequences with partially overlapping deletions, most MSA programs preferentially aligned the gaps vertically at the expense of incorrectly aligning residues in the flanking regions. Of the programs assessed, only DIALIGN-T was able to place overlapping gaps correctly relative to one another, but this was usually context dependent and was observed only in some of the data sets. In data sets containing sequences with non-overlapping deletions, both DIALIGN-T and MAFFT (G-INS-I) were able to align gaps with near-perfect accuracy, but only MAFFT produced the correct alignment consistently. The same was true for data sets that comprised isoforms of alternatively spliced gene products: both DIALIGN-T and MAFFT produced highly accurate alignments, with MAFFT being the more consistent of the 2 programs. Other programs, notably T-COFFEE and ClustalW, were less accurate. For all data sets, alignments produced by different MSA programs differed markedly, indicating that reliance on a single MSA program may give misleading results. It is therefore advisable to use more than one MSA program when dealing with sequences that may contain deletions or insertions, particularly for high-throughput and pipeline applications where manual refinement of each alignment is not practicable.     

用于聚合酶链式反应(PCR)引物设计的计算机程序

 本文报道了两个用于PCR引物设计的计算机程序PCRDESN和PCRDESNA。PCRDESN程序主要从以下4个方面评价用户自己设计的一对引物的质量:(1)引物内的碱基反向重复或发夹结构,(2)两个引物之间的碱基互补配对,(3)两个引物之间的同源性,(4)引物的碱基组成及特点和T_m值计算。通过用多例文献发表的及本院有关实验室提供的引物对序列的验证,确定了程序的运算参数,证明该程序能较好地检验引物对的质量和解释某些PCR实验失败的原因。PCRDESNA程序采用逐级优化的方法和比PCRDESN所选用的更严紧的引物选择参数对用户提供的核酸序列进行快速检索,以确定所有可能的和合适的引物对。     

MUST, a computer package of Management Utilities for Sequences and Trees.

The MUST package is a phylogenetically oriented set of programs for data management and display, allowing one to handle both raw data (sequences) and results (trees, number of steps, bootstrap proportions). It is complementary to the main available software for phylogenetic analysis (PHYLIP, PAUP, HENNING86, CLUSTAL) with which it is fully compatible. The first part of MUST consists of the acquisition of new sequences, their storage, modification, and checking of sequence integrity in files of aligned sequences. In order to improve alignment, an editor function for aligned sequences offers numerous options, such as selection of subsets of sequences, display of consensus sequences, and search for similarities over small sequence fragments. For phylogenetic reconstruction, the choice of species and portions of sequences to be analyzed is easy and very rapid, permitting fast testing of numerous combinations of sequences and taxa. The resulting files can be formatted for most programs of tree construction. An interactive tree-display program recovers the output of all these programs. Finally, various modules allow an in-depth analysis of results, such as comparison of distance matrices, variation of bootstrap proportions with respect to various parameters or comparison of the number of steps per position. All presently available complete sequences of 28S rRNA are furnished aligned in the package. MUST therefore allows the management of all the operations required for phylogenetic reconstructions.     

TREECON: A software package for the construction and drawing of evolutionary trees

A package of programs (run by a management program called TREECON)was developed for the construction and drawing of evolutionarytrees. The program MATRIX calculates dissimilarity values andcan perform bootstrap analysis on nucleic acid sequences. TREEimplements different evolutionary tree constructing methodsbased on distance matrices. Because some of these methods produceunrooted evolutionary trees, a program ROOT places a root onthe tree. Finally, the program DRAW draws the evolutionary tree,changes its size or topology, and produces drawings suitablefor publication. Whereas MATRIX is suited only for nucleic acids,the modules TREE, ROOT and DRAW are applicable to any kind ofdissimilarity matrix. The programs run on IBM-compatible microcomputersusing the DOS operating system.     

DePIE: Designing Primers for Protein Interaction Experiments

Several primer prediction and analysis programs have been developed for diverse applications. However, none of these existing programs can be directly used for the design of primers in protein interaction experiments, since proteins may have transmembrane domains (TMDs) and/or a signal peptide that must be excluded from experiments. Furthermore, it is frequently the case that a short restriction sequences must be added to each primer in order to clone PCR products into a given destination vectors for expression. DePIE, a web-based primer design tool, was developed to address these deficiencies. The program takes as input NCBI protein accession numbers and returns primer information including nucleotide sequences, thermodynamic melting temperature of the nucleotide sequences and the target positions. DePIE is implemented in JAVA, PERL and PHP and has proven to be very efficient in designing primers for our interaction experiments. DePIE services can be accessed at the web site: http://biocore.unl.edu/primer/primerPI.html.     

Exon discovery by genomic sequence alignment

MOTIVATION: During evolution, functional regions in genomic sequences tend to be more highly conserved than randomly mutating 'junk DNA' so local sequence similarity often indicates biological functionality. This fact can be used to identify functional elements in large eukaryotic DNA sequences by cross-species sequence comparison. In recent years, several gene-prediction methods have been proposed that work by comparing anonymous genomic sequences, for example from human and mouse. The main advantage of these methods is that they are based on simple and generally applicable measures of (local) sequence similarity; unlike standard gene-finding approaches they do not depend on species-specific training data or on the presence of cognate genes in data bases. As all comparative sequence-analysis methods, the new comparative gene-finding approaches critically rely on the quality of the underlying sequence alignments. RESULTS: Herein, we describe a new implementation of the sequence-alignment program DIALIGN that has been developed for alignment of large genomic sequences. We compare our method to the alignment programs PipMaker, WABA and BLAST and we show that local similarities identified by these programs are highly correlated to protein-coding regions. In our test runs, PipMaker was the most sensitive method while DIALIGN was most specific. AVAILABILITY: The program is downloadable from the DIALIGN home page at http://bibiserv.techfak.uni-bielefeld.de/dialign/.     

Two accurate sequence,structure, and phylogenetic template-based RNA alignment systems

Background

The analysis of RNA sequences, once a small niche field for a small collection of scientists whose primary emphasis was the structure and function of a few RNA molecules, has grown most significantly with the realizations that 1) RNA is implicated in many more functions within the cell, and 2) the analysis of ribosomal RNA sequences is revealing more about the microbial ecology within all biological and environmental systems. The accurate and rapid alignment of these RNA sequences is essential to decipher the maximum amount of information from this data.

Methods

Two computer systems that utilize the Gutell lab's RNA Comparative Analysis Database (rCAD) were developed to align sequences to an existing template alignment available at the Gutell lab's Comparative RNA Web (CRW) Site. Multiple dimensions of cross-indexed information are contained within the relational database - rCAD, including sequence alignments, the NCBI phylogenetic tree, and comparative secondary structure information for each aligned sequence. The first program, CRWAlign-1 creates a phylogenetic-based sequence profile for each column in the alignment. The second program, CRWAlign-2 creates a profile based on phylogenetic, secondary structure, and sequence information. Both programs utilize their profiles to align new sequences into the template alignment.

Results

The accuracies of the two CRWAlign programs were compared with the best template-based rRNA alignment programs and the best de-novo alignment programs. We have compared our programs with a total of eight alternative alignment methods on different sets of 16S rRNA alignments with sequence percent identities ranging from 50% to 100%. Both CRWAlign programs were superior to these other programs in accuracy and speed.

Conclusions

Both CRWAlign programs can be used to align the very extensive amount of RNA sequencing that is generated due to the rapid next-generation sequencing technology. This latter technology is augmenting the new paradigm that RNA is intimately implicated in a significant number of functions within the cell. In addition, the use of bacterial 16S rRNA sequencing in the identification of the microbiome in many different environmental systems creates a need for rapid and highly accurate alignment of bacterial 16S rRNA sequences.

     

MITE-Hunter: a program for discovering miniature inverted-repeat transposable elements from genomic sequences

Miniature inverted-repeat transposable elements (MITEs) are a special type of Class 2 non-autonomous transposable element (TE) that are abundant in the non-coding regions of the genes of many plant and animal species. The accurate identification of MITEs has been a challenge for existing programs because they lack coding sequences and, as such, evolve very rapidly. Because of their importance to gene and genome evolution, we developed MITE-Hunter, a program pipeline that can identify MITEs as well as other small Class 2 non-autonomous TEs from genomic DNA data sets. The output of MITE-Hunter is composed of consensus TE sequences grouped into families that can be used as a library file for homology-based TE detection programs such as RepeatMasker. MITE-Hunter was evaluated by searching the rice genomic database and comparing the output with known rice TEs. It discovered most of the previously reported rice MITEs (97.6%), and found sixteen new elements. MITE-Hunter was also compared with two other MITE discovery programs, FINDMITE and MUST. Unlike MITE-Hunter, neither of these programs can search large genomic data sets including whole genome sequences. More importantly, MITE-Hunter is significantly more accurate than either FINDMITE or MUST as the vast majority of their outputs are false-positives.     

The current status and portability of our sequence handling software.

I describe the current status of our sequence analysis software. The package contains a comprehensive suite of programs for managing large shotgun sequencing projects, a program containing 61 functions for analysing single sequences and a program for comparing pairs of sequences for similarity. The programs that have been described before have been improved by the addition of new functions and by being made very much easier to use. The major interactive programs have 125 pages of online help available from within them. Several new programs are described including screen editing of aligned gel readings for shotgun sequencing projects; a method to highlight errors in aligned gel readings, new methods for searching for putative signals in sequences. We use the programs on a VAX computer but the whole package has been rewritten to make it easy to transport it to other machines. I believe the programs will now run on any machine with a FORTRAN77 compiler and sufficient memory. We are currently putting the programs onto an IBM PC XT/AT and another micro running under UNIX.