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61
Documentation / AffinityProfile
« Last post by xiangjun on September 29, 2016, 12:54:18 pm »
AffinityProfile is designed to scan a sequence file against a list of PSAMs or IUPAC motifs to get single base resolution affinity profiles. For convenience, it also allows for a single PSAM (-psam=one_PSAM_file) or an IUPAC motif (-motif=one_IUPAC_motif) to be specified directly on the command line. The PSAMs can be from a MatrixREDUCE or MotifREDUCE run, or a collection of pseudo-PSAMs from literature (as in $REDUCE_SUITE/data/PSAMs/ directory).

AffinityProfile [options] -sequence=seqfile \
                         -psam=one_PSAM_file | -psam_list=list_of_PSAMs |
                         -motif=one_IUPAC_motif | -motif_list=list_of_motifs

  Required parameters:
    -sequence=file_name  --- name of sequence file in FASTA format
    -psam=one_PSAM_file    --- file name of one PSAM
    -psam_list=list_of_PSAMs --- file name containing a list of PSAMs
    -motif=IUPAC_motif     --- one IUPAC motif
    -motif_list=list_of_motifs --- file name containing a list of IUPAC motifs

  Optional parameters:
    [-threshold=float]   --- threshold of affinity for output (0.0)
    [-output=dir_name]   --- path to the output directory (./)
    [-prefix=string]     --- prepended to output profile name (aff_)
    [-affsum=string]     --- file of total affinity per sequence (seq_psam.dat)
    [-detail]            --- also output detailed affinity along each sequence
    [-ids=string]        --- a ',' or ';' delimited list of IDs
    [-column]            --- used with -ids, set profile column-wise for each id
    [-normalize]         --- linear re-scale (per PSAM) the maximum profile to 1.0
    [-strand=integer]      ---  1 |+1 |F | L for leading strand;
                                2 |+2 |B     for both strands;
                               -1 | R |C     for reverse complementary;
                                   with -motif, default to leading strand
                                   with -psam, default to PSAM setting

  Usage:
      (1) AffinityProfile -sequence=$REDUCE_SUITE/data/sequence/YeastUpstream.fasta \
                          -psam_list=psams.list
      (2) AffinityProfile -sequence=$REDUCE_SUITE/data/sequence/YeastUpstream.fasta \
                          -motif=aaaccct
      (3) AffinityProfile -sequence=$REDUCE_SUITE/data/sequence/YeastUpstream.fasta \
                          -motif=aaaga -ids='YAL001C;YAL002W' -column


Notes:

  • By default, the reported affinities take into account of the threshold (default to 0) specified on the command-line. Thus, per slide window, if the affinity is less than the threshold, it will neither be outputted per window nor added into the sum per sequence (as in file seq_psam_thr.dat, see below).
  • AffinityProfile also outputs two files with fixed names: seq_psam.dat (which sums up all affinities, even those below threshold, for reference and comparison) and seq_psam_thr.dat (taking into account of threshold) that contain the sum of affinities per sequence in a tab-delimited format that can be fed directly into Transfactivity.
  • Following each run of MatrixREDUCE or MotifREDUCE, two fix-named files, psams.list and motifs.list, are also available, which can be fed into AffinityProfile, as shown in the first example above.
  • This is yet another example illustrating how the REDUCE Suite has been designed with tools that are seamlessly inter-connected to allow for great flexibility.
62
Documentation / OptimizePSAM
« Last post by xiangjun on September 29, 2016, 12:41:18 pm »
This program performs a single point optimization of either an initial (pseudo-) PSAM or a seed motif against the measurement file and sequence. Internally, it uses exactly the same Levenberg-Marquardt non-linear least squares fitting algorithm as in MatrixREDUCE.

OptimizePSAM [options] -sequence=seqfile -measurement=measfile \
                       -psam=PSAM_file | -motif=IUPAC_Motif

  Required parameters:
    -sequence=file_name    --- name of a FASTA sequence file
    -meas=measfile         --- measurement (expression/binding) file in tab-delimited format
    -psam=PSAM_file        --- PSAM file to be optimized
    -motif=seed_motif      --- Seed IUPAC motif sequence to be optimized

  Optional parameters:
    [-output=dir_name]     --- path to the output directory (./)
    [-p_value=float]       --- threshold to decide if optimized PSAM is significant (0.001)
    [-filename=file]       --- name of the optimized PSAM
    [-strand=integer]      ---  1 |+1 |F | L for leading strand (1);
                                2 |+2 |B     for both strands;
                               -1 | R |C     for reverse complementary;
                                0 | A |D     auto-detection (check 1 and 2)
    [-runlog=[stderr|stdout|file]]
                           --- direct running diagnostics message to stderr,
                               stdout or a file (stderr)
    [-help]                --- print out this help message

  Usage:
    OptimizePSAM \
       -measurement=$REDUCE_SUITE/data/mRNA_expression/Spellman1998AlphaTimeCourse.tsv \
       -sequence=$REDUCE_SUITE/data/sequence/YeastUpstream.fasta \
       -motif=ACGCGT -file=ACGCGT.psam


Notes:

  • In PSAM format, the initial seed motif ACGCGT is expressed as follows:
    # A            C            G            T             # no. opt
    # +============+============+============+============ # ==+===+==
      1            0            0            0             #   1   A
      0            1            0            0             #   2   C
      0            0            1            0             #   3   G
      0            1            0            0             #   4   C
      0            0            1            0             #   5   G
      0            0            0            1             #   6   T

  • The optimized PSAM in file ACGCGT.psam is as follows. Note specifically the changes of Ws from 1s and 0s of initial sequence motif (above) to some fractions with a maximum of 1 in each position in the optimized PSAM.
    # A            C            G            T             # no. opt
    # +============+============+============+============ # ==+===+==
      1            0.143386     0.156974     0.332267      #   1   A
      2.38995e-06  1            0.133257     6.623e-18     #   2   C
      0.203947     0.0136109    1            6.43632e-11   #   3   G
      3.39323e-14  1            4.38946e-14  3.19148e-17   #   4   C
      0.0655988    0.122631     1            1.13119e-13   #   5   G
      0.422826     0.221149     0.182984     1             #   6   T

  • As shown in the following diagnostic message from running OptimizePSAM, this optimization step increases the fitted R2 from 0.0414328 to 0.0552883, and the PSAM is significant.
    Best seed experiment:
       number of tested candidate experiments: 18
       intercept: coef=-0.12248   t-value=-18.4713   p-value=5.77026e-74
       slope:     coef=+0.363975   t-value=+15.4353   p-value=1.18198e-52
       r2=0.0414328   SSY=1323.85   SSE=1269   SSR=54.8506
       matches[matched-ids/total-ids]: 348[307/5514]   experiment: alpha_factor_release_sample016 [4]
           and of sequence on forward strand
    Optimizing:
         20 (1250.65): converged with gradient: 0.0349271 <= 0.05
    PSAM linear fit statistics:
       intercept: coef=-0.186363   t-value=-23.1987   p-value=1.12291e-113
       slope:     coef=+0.325095   t-value=+17.9606   p-value=3.83258e-70
       r2=0.0552883   SSY=1323.85   SSE=1250.65   SSR=73.1932
    Checking PSAM significance:
       |r|=0.235135   r0=0.0688157   sigma=0.00888813   t_value=18.7125
       E-value=4.19638e-76
       This PSAM is significant (E-value smaller than specific cutoff of 0.001)

63
Documentation / HTMLSummary
« Last post by xiangjun on September 27, 2016, 02:51:44 pm »
Following a MatrixREDUCE or MotifREDUCE run, a bunch of output files (model parameters, PSAMs etc.) are generated. The HTMLSummary utility program is provided to summarize the main results in a intuitive, easy to follow HTML page so that (even non-expert) users can quickly make sense of their findings, e.g., the top list of significant PSAMs (motifs). Internally, HTMLSummary makes system-calls to the utility program LogoGenerator to create the logo images. These inter-connected programs, plus a few others, constitute the REDUCE Suite.

HTMLSummary [options] [-file=HTMLFile]

  Required parameters:
    none

  Optional parameters:
    [-output=dir_name]  --- path to an MatrixREDUCE/MotifREDUCE run directory,
                            used both as input and output for HTML summary (./)
    [-copy]             --- copy CSS, JavsScript and associated image files to
                            the output directory to make the HTML self-contained
    [-rc]               --- logo based on reverse complementary strand
    [-width=ThumbnailImageWidthInPixel]   (145)
    [-height=ThumbnailImageHeightInPixel] (90)
    [-psam_list=list_of_PSAMs] --- generate a summary LOGO page for the list
                                   of PSAMs in the file
    [-file=HTMLFile]    --- HTML output file name (index.html)

  Usage: (following a MatrixREDUCE run)
    HTMLSummary
    HTMLSummary -psam=psams.list -file=psams.html
64
Documentation / MatrixREDUCE
« Last post by xiangjun on September 27, 2016, 02:43:34 pm »
MatrixREDUCE uses genome-wide occupancy data for a transcription factor (e.g. ChIP-chip or mRNA expression data) and associated nucleotide sequences to discover the sequence-specific binding affinity of the transcription factor. The sequence specificity of the transcription factor's DNA-binding domain is modeled using a position-specific affinity matrix (PSAM), representing the change in the binding affinity (Kd) whenever a specific position within a reference binding sequence is mutated.

MatrixREDUCE [options] -sequence=seqfile -measurement=measfile

  Required parameters:
    -sequence=seqfile     --- sequence file in FASTA format
    -meas=measfile        --- measurement (expression/binding) file in tab-delimited format

  Optional parameters:
    [-topo_list=topofile]  --- name of topology file (up_to_octamers)
    [-topo=topology]       --- single topology pattern, e.g., X3--X4
    [-multifit]            --- switch to seed/optimize using all experiments
                                   [added based on code from Pilar -- thanks!]
    [-dicfile=file]        --- list of motifs to check against. IUPAC wild cards
                                   allowed; no length limit
    [-ntop=integer]        --- number of top seed motifs to print out (10)
    [-iupac_pos=integer]   --- number of positions to check for IUPAC degeneracy (0)
    [-iupac_sym=string]    --- IUPAC symbols to check against ('KMRSWYBDHVN')

    [-output=dir_name]     --- path to the output directory (./)
    [-p_value=float]       --- threshold to stop looking for new PSAMs (0.001)
    [-max_motif=integer]   --- maximum # of PSAMs to search (20)
    [-strand=integer]      ---  1 |+1 |F | L for leading strand;
                                2 |+2 |B     for both strands;
                               -1 | R |C     for reverse complementary;
                                0 | A |D     auto-detection (check 1 and 2)

    [-runlog=[stderr|stdout|file]]
                           --- direct running diagnostics message to stderr,
                                   stdout or a specific file (stderr)
    [-help]                --- print out this help message

  Usages:
    mkdir -p results
    MatrixREDUCE \
       -meas=$REDUCE_SUITE/data/mRNA_expression/Spellman1998AlphaTimeCourse.tsv \
       -sequence=$REDUCE_SUITE/data/sequence/YeastUpstream.fasta \
       -topo_list=$REDUCE_SUITE/data/topology/up_to_octamers -o=results
    HTMLSummary -o=results

    mkdir -p X6
    MatrixREDUCE \
       -meas=$REDUCE_SUITE/data/mRNA_expression/Spellman1998AlphaTimeCourse.tsv \
       -sequence=$REDUCE_SUITE/data/sequence/YeastUpstream.fasta \
       -topo=X6 -o=X6
    HTMLSummary -c -o=X6

65
Documentation / MotifREDUCE
« Last post by xiangjun on September 27, 2016, 02:39:42 pm »
REDUCE is an acronym that stands for Regulatory Element Detection Using Correlation with Expression. Based on a simple model for transcriptional regulation by independently acting transcription factors, REDUCE makes it possible to find regulatory elements based on a single microarray experiment. MotifREDUCE in the REDUCE Suite is a more robust and efficient reimplementation of the "original REDUCE algorithm" by Bussemaker et al (2001).

MotifREDUCE [options] -sequence=seqfile -measurement=measfile

  Required parameters:
    -sequence=seqfile     --- sequence file in FASTA format
    -meas=measfile        --- measurement (expression/binding) file in tab-delimited format

  Optional parameters:
    [-topo_list=topofile]  --- name of topology file (up_to_octamers)
    [-topo=topology]       --- single topology pattern, e.g., X3--X4
    [-dicfile=file]        --- list of motifs to check against. IUPAC wild cards
                                   allowed; no length limit
    [-ntop=integer]        --- number of top seed motifs to print out (10)
    [-iupac_pos=integer]   --- number of positions to check for IUPAC degeneracy (0)
    [-iupac_sym=string]    --- IUPAC symbols to check against ('KMRSWYBDHVN')

    [-output=dir_name]     --- path to the output directory (./)
    [-p_value=float]       --- threshold to stop looking for new motifs (0.001)
    [-max_motif=integer]   --- maximum # of motifs to search (20)
    [-strand=integer]      ---  1 |+1 |F | L for leading strand;
                                2 |+2 |B     for both strands;
                               -1 | R |C     for reverse complementary;
                                0 | A |D     auto-detection (check 1 and 2)

    [-runlog=[stderr|stdout|file]]
                           --- direct running diagnostics message to stderr,
                                   stdout or a specific file (stderr)
    [-help]                --- print out this help message

  Usage:
    mkdir -p results   # use topology file (up_to_heptamers)
    MotifREDUCE \
        -meas=$REDUCE_SUITE/examples/MotifREDUCE/yeast_sample.csv \
        -sequence=$REDUCE_SUITE/examples/MotifREDUCE/genome5pns600.fasta \
        -topo_list=$REDUCE_SUITE/examples/MotifREDUCE/up_to_heptamers \
        -o=results
    HTMLSummary -c -o=results

Notes:
  • The command-line user-interface of MotifREDUCE is identical to that of MatrixREDUCE, but skips the Levenberg-Marquardt non-linear least-squares optimization of weight matrix (Ws). The result is a list of motifs, which are expressed in matrix form with 1s and 0s.
  • The above example dataset takes ~10s and finds 10 significant motifs on a contemporary laptop computer.
66
General Discussion / Re: Generate RNA logos with LogoGenerator
« Last post by knie on September 27, 2016, 02:01:22 am »
Hi Xiang-Jun,

Got it! And thanks a lot for the information about logo colors :D

Best,
Kate
67
General Discussion / Re: Generate RNA logos with LogoGenerator
« Last post by xiangjun on September 26, 2016, 11:18:16 pm »
Hi Kate,

Yes. You need to edit file $REDUCE_SUITE/html/LogoGenerator_PS.def slightly, as shown below.

Current (default settings):
Code: [Select]
/colorDict <<
    (A) green       (a) m_green
    (C) blue        (c) m_blue
    (G) orange      (g) m_orange
    (T) red         (t) m_red
    (U) cyan        (u) m_cyan
    (X) white
>> def

Change (U) cyan to (U) red will achieve what you asked. Of course, you can completely redefine the coloring scheme as you see fit in section "default color set".

A bit background info for the default colors: the A green, C blue, G orange, and T red coloring scheme follows WebLogo for DNA. On the other hand, the 3DNA software for 3D structures of DNA/RNA uses another convention: A red, C yellow, G green, T blue, and U cyan. See http://x3dna.org/articles/seeing-is-understanding-as-well-as-believing for some examples. So I ended up with cyan for U in RNA logo. Moreover, the REDUCE Suite Forum is based on the 3DNA Forum.

Best regards,

Xiang-Jun
68
General Discussion / Re: Generate RNA logos with LogoGenerator
« Last post by knie on September 26, 2016, 06:03:44 pm »
Hi Xiang-Jun,

As a followup question:
The default color of the nucleotide "U" from `LogoGenerator -rna` is light blue. Is it possible to change it to red (like the color of "T" in DNA logos)?

Best,
Kate
69
General Discussion / Re: Generate RNA logos with LogoGenerator
« Last post by knie on September 22, 2016, 03:31:21 pm »
Hi Xiangjun,

Perfect! Many thanks for the prompt reply.

Best,
Kate
70
General Discussion / Re: Generate RNA logos with LogoGenerator
« Last post by xiangjun on September 22, 2016, 10:29:37 am »
Hi Kate,

Thanks for using the REDUCE Suite and for posting your question on the Forum.

Yes, LogoGenerator is applicable to RNA as well as DNA. You just need to specify an additional -rna option on the command-line, and everything else should work as expected. Please have a try and report back any issues you may have.

Best regards,

Xiang-Jun


PS. The REDUCE Suite has more features than those documented (with the command-line "-h" option). The default settings are designed to work for its advertised purpose only.
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Created and maintained by Dr. Xiang-Jun Lu [律祥俊]. See also http://forum.x3dna.org and http://x3dna.org