HELP AND DOCUMENTATION

NAMEThis field is necessary. A default name `query' is given in case client does not wish to give his/her own name to the query.Paste your sequenceThe DNA sequence can be pasted into the text area. In case the Format of the sequence is any of the standard ones (EMBL, FASTA, GENBANK, etc.) then `INPUT-FORMAT' should be selected to `FORMATTED' or to `NON-FORMATTED' in case the input sequence is just nucleotide sequence.UPLOAD-FILEFile containing nucleotide sequence in any of the standard formats or a non-formatted DNA sequence can be uploaded using this option. Users can- -not upload a sequence file and paste the sequence simultaneously to be analyzed together.INPUT-FORMATThe program recognizes any of the standard formats (EMBL, FASTA, GENBANK, etc.). It uses the ReadSeq program developed by Dr. Don Gilbert, Biology Dept., Indiana University, to read the input sequence and can accept most commonly used standard sequence formats. Users should select `FORMATTED' if the input sequence is in any of these standard formats or `NON-FORMAT- -TED' if the input sequence is just the nucleotide sequence.OUTPUT-FORMATThe server gives the result in either of the two formats-TABULARorGRAPHICALIn TABULAR OUTPUT it gives the values of the Fourier spectra or Power at different frequencies in case of GENESCAN or m1, m2, m3 in case of ZCurve. In GRAPHICAL OUTPUT it gives the plot of Power vs frequency in case of GENESCAN while in case of ZCurve it is a plot of m1 vs m3. In case of FTG the TABULAR OUTPUT is a table of Power at different frequencies while the GRAPHICAL OUTPUT gives a spectrum of Power vs frequency.FROM_&_TOUsers can select a particular region from the input sequence data to anal- -yze through this property. Certain tips-- If the user wants to do a complete sequence analysis but does not know the last base number, he/she can leave the `TO' field empty and give `1' in the `FROM' field. If the user wants to do analysis on the region 230-590 for eg. he/she should give `230' in the `FROM' field and `590' in the `TO' field. Do not leave `FROM' field empty.ALGORITHMBasically there are three different algorithms and their three modificat- -ions. The Algorithms are -GENESCAN, LENGTHEN-SHUFFLE and FTG while their modifications are- GENESCAN-WINDOW, LENGTHEN-SHUFFLE-WINDOW and FTG-WINDOW. The WINDOW options of each of these algorithms are given for convenience. A long DNA sequence to be analyzed for multiple protein-coding regions can be analyzed using these options. Once protein coding regions are identified they can be confirmed for periodicity using the original versions of the algorithms.GENESCANThis algorithm uses a Fourier technique based on a distinctive feature of protein-coding regions, the 3-base periodicity. The signature of this (also other) periodicity can be observed most directly through the Fourier analysis. A sequence of N nucleotides may be formally viewed as a symbol string, {xj, j=1,2,.....,N}, where xj is one of the four symbols A, T, G and C, and denotes the occurrence of that particular nucleotide in position j. One can define a binary indicator function or projection operator Ua which selects the elements of the sequence that are equal to the symbol a, namely Ua(xj)=1 if xj is a and 0 otherwise. Using the operators UA,UT, UG,UC, successively on a DNA sequence yields four binary sequences, as illustrated below; Sequence GGATACACTTTAGAG Apply UA 001010100001010 Apply UT 000100001110000 Apply UG 110000000000101 Apply UC 000001010000000 Figure 1. Thus, any DNA sequence can be converted to four binary sequences, which can then be Fourier analyzed in the normal manner, to examine correlat- ions between the symbols. The total Fourier spectrum of the DNA sequence is the sum of these individual spectra, namely; -----------(1) where the discrete frequency f=k/N, with k=1,2,....N/2. Sa(f) is the par- -tial spectrum corresponding to the symbol a=A, G, C, or T. The average of the total spectrum, S^, can be calculated from the frequency of occur- -rence, þa of each symbol (a=A, T, G, C) as; -----------(2) For protein-coding sequences from a variety of organisms, the Fourier sp- -ectrum [equation(1)] reveals the characteristic periodicity of three as a distinct peak at frequency f=1/3. No such `peak' above noise level is apparent for non-protein coding sequences such as rRNA, intergenic spacers and introns, which have a flat Fourier spectrum devoid of any periodicity. In order to contrast signal-to-noise ratio of the peak at f=1/3, is given as; -----------(3) P=4 is used as discriminator between coding and non-coding sequences. For a detailed description of the algorithm please refer the original paper (Tiwariet al., 1997). The academic version of the program is available for distribution and can be accessed at http://202.41.10.146/GS.htmlLENGTHEN-SHUFFLEDue to the limited length (usually 100bp or so) of the window used in gene finding process, the application of the Fourier measure is without imp- -ressive success. For a longer sequence, >1024bp, it is easier to detect the periodicity by the FFT algorithm. This algorithm find a way to solve this problem. FORMAT OF Z CURVES: Consider a DNA sequence with N bases read from the 5-end to the 3-end. Begining from th first base, inspect the sequence one base at a time. Let the number of steps be denoted by n, i.e. n=1,2,....N. In the nth step, count the cumulative numbers of the bases A, C, G and T, respectively, occurring in the subsequence from the first to the nth base in the DNA sequence inspected. Denote the four positive integers by An, Cn, Gn, and Tn, respectively. The Zcurve consists of a series of nodes Pn(n=1,2,....N) whose coordinates are denoted by xn, yn, and zn. It was shown that xn = 2(An + Gn) - n, yn = 2(An + Cn) - n, n=0,1,2...........,N -----------(4) zn = 2(An + Tn) - n, where A0=C0=G0=T0=0 and thus x0=y0=z0=0. The connection of nodes P0(i.e. the origin), P1, P2,...PN one by one by lines is defined as the Z curve of the DNA sequence inspected. We then define; §xn = xn - xn-1, §yn = yn - yn-1, n=1,2,....N ------------(5) §zn = zn - zn-1, where §xn, §yn and §zn can only have the values of 1 or -1. §xn is equal to 1 when the nth base is A or G(Purine), or -1 when the nth base is C or T(Pyrimidine); §yn is equal to 1 when the nth base is A or C(amino-type), or -1 when the nth base is G or T(keto-type); §zn is equal to 1 when the nth base is A or T(weak hydrogen bond), or -1 when the nth base is G or C (strong hydrogen bond). Therefore, DNA sequence can be decomposed into 3 series of digital signals, consisting of 1 or -1, each of which has clear biological meaning. The first series of digital signals §xn represents the distribution of the bases of the purine/pyrimidine along the DNA sequences. The second series §yn represents the distribution of the bases of the amino/keto types along the sequence. Similarly, the third series §zn, represents the distribution of the bases of the strong/weak hydrogen bonds along the sequence. A LENTHEN-SHUFFLE FOURIER TRANSFORM: The relatively short DNA sequence D(<150bp) is first lengthened by repeating the sequence K times, where K=1200/D. Obviously a bogus periodicity of D will be observed in the power spectrum of the FFT. To eliminate such a bogus periodicity, and at the same time keep the periodicity of 3 uncha- -nged, the lengthened sequence is then shuffled M times with three cons- -ecutive bases as a unit. A typical value of M used here is 10,000. As mentioned above, based on the the format of the Zcurve, any DNA sequ- -ence can be transformed into three series of digital signals, §xn, §yn, and §zn, to which to apply the FFT algorithm. The power spectrum for each series is calculated as follows: -------------(6) where PC(f) is the power spectrum associated with §Cn which represents §xn, §yn and §zn. Now three values are obtained, m1=Px(N/3), m2=Py(N/3) and m3=Pz(N/3). The Fisher linear discriminant equation is used for making the coding/non-coding decision. For a detailed description of the algorithm please refer the original paper (Yanet al., 1998). The program is available on request from C.-T. Zhang ctzhang@tju.edu.cnFTGFTG algorithm combines the properties of both GENESCAN and LENGTHEN-SHUF- -FLE algorithms to improve the accuracy of gene prediction. The drawback of the GENESCAN algorithm is that the spectrum of any short DNA sequence say <150bp, a window-size generally used, is not clear thereby the peri- -odicity becomes incoherent. Though the LENGTHEN-SHUFFLE algorithm tries to address this problem, it has the drawback of not indicating the type of periodicity that a DNA sequence has. FTG tries to overcome these limi- -tations by combining the essential parts of the two algorithms- GENESCAN, and LENGTHEN-SHUFFLE so as to amplify the three-base periodicity of the DNA sequence. FTG takes short nucleotide sequences of length D (where D<=1200) and then amplifies it by repeating it K times (where K=(1200/D)+1). The bogus per- -iodicity of D is removed by shuffling the sequence M times (where M=10000) Now the extended DNA sequence is considered as a symbol string, {xj, j=1,2 ....,N}, where xj is one of the four symbols A, C, G and T, and denotes the occurrence of that particular nucleotide in position j. Now defined a binary indicator function or projection operator Ua which selects the elements of the sequence that are equal to the symbol a, namely Ua(xj)=1 if xj is a and 0 otherwise. Using the operators UA, UT, UG and UC successively on a DNA sequence yields four binary sequence, as illustrated in Figure 1. The four binary sequences obtained can then be Fourier analyzed in the normal manner. Fourier spectrum of the DNA sequence is calculated using equation 1, while the average spectrum is calculated by using equation 2. Peak at f=1/3 is obtained and signal-to-noise ratio of the peak at f=1/3 is obtained using equation 3.GENESCAN-WINDOW, LENGTHEN-SHUFFLE-WINDOW and FTG-WINDOWThe user can analyze long DNA sequences using these WINDOW option of each algorithm. User has to specify Step-size and Window length for these three algorithm. The program takes overlapping Window separated by the Step-size and analyzes each Window and outputs the values for that Window. The adva- -tage that this option offers is to analyze the whole input sequence con- -tinuously window-by-window, saving precious time.Step-Size_&_WindowConsider a DNA sequence of 10000 bp length. Now I want to analyze the sequence with overlapping windows of 150 bp which are overlapping each other. Suppose I want to take window after every 5 bp. Then I would give a Step-size of 5 and Window of 150 in the submission form. 1--ACGTGCTAGCTGATGCTAGTGC---100--CATCGACTAGCATCAGCTACAGCTACGATCAGCACTGATC----10000|----------------------|<-------------Window-length----1st Window--------->|----------------------|<-------------2nd Window-------------------------->|--|Step-size|----------------------|<------------------3rd Window---------------------> It is useful to remember that results will be much more reliable for a smaller Step-size.TABULAR_OUTPUTGENESCANThe tabular output option for GENESCAN outputs Power for different frequencies. The average of the Peaks for a spectrum is also output along with Peak-to-noise ratio at f=1/3. Trivia such as nucleotide composition and dinucleotide content are also computed by the program.LENGTHEN-SHUFFLEThe LENGTHEN-SHUFFLE algorithm outputs m1, m2, m3 as table along with the sequence composition analysis report.FTGThe tabular output option for FTG outputs Peaks at different freque- -ncies. The average of the Peaks for a spectrum is also output along with Peak-to-noise ratio at f=1/3. Nucleotide composition and dinuc- -leotide content are also computed by the program.GENESCAN-WINDOWFor each window analyzed the GENESCAN-WINDOW option outputs the Peak or Power at f=1/3. The start-point in the table refers to the position of first base of the window. Normal sequence analysis of the DNA seq- -uence is also given.LENGTHEN-SHUFFLE-WINDOWFor each window of the DNA sequence m1, m2, and m3 are computed and output by the program for this option. Position in this table refers to the first base position of the window in the input DNA sequence.FTG-WINDOWThe position in the table refers to the first base of the window and the corresponding Power refers to the Peak at f=1/3 for that window. Normal nucleotide compositional analysis is also given.GRAPHICAL_OUTPUTGENESCANA plot of Power[S(f)] vs frequency(f) is output with this option. The periodicity of three can be visible as a peak at f=0.33(1/3). Average peak of the spectrum and Peak at f=1/3 is given. A periodicity of 10 can be visible as a peak at f=0.10(1/10). Similarly other periodicity can be observed with this plot.LENGTHEN-SHUFFLEPlots of m3 vs m1, m3 vs m2, m2 vs m1, are output with this option.FTGThis plot is quiet similar to the plot obtained from GENESCAN option except that FTG option works best for short nucleotide sequences.GENESCAN-WINDOWThe Window version of the GENESCAN algorithm gives a plot of Peak at f=1/3 for each window vs sequence length. The red horizontal line is the default threshold for the coding/non-coding decision. If the line of the spectrum goes above this threshold then the region is considered coding.LENGTHEN-SHUFFLE-WINDOWThree graphs of sequence length vs m1, m2, and m3, respectively are given. In addition a plot of m3 vs m1 for different window is given.FTG-WINDOWThis graph is quiet similar to that of GENESCAN-WINDOW graph except it gives a plot of Power at f=1/3 for overlapping windows of size less less than 1200 (ideally less than 150).