Highly customizable, ambiguity-aware dotplots for visual sequence analyses
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README.md

FlexiDot: Highly customizable, ambiguity-aware dotplots for visual sequence analyses

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FlexiDot is a cross-platform dotplot suite generating high quality self, pairwise and all-against-all visualizations. To improve dotplot suitability for comparison of consensus and error-prone sequences, FlexiDot harbors routines for strict and relaxed handling of mismatches and ambiguous residues. The custom shading modules facilitate dotplot interpretation and motif identification by adding information on sequence annotations and sequence similarities to the images. Combined with collage-like outputs, FlexiDot supports simultaneous visual screening of a large sequence sets, allowing dotplot use for routine screening.

Citation

If you use FlexiDot in your research, please cite us:

Kathrin M. Seibt, Thomas Schmidt, and Tony Heitkam (2018) "FlexiDot: Highly customizable, ambiguity-aware dotplots for visual sequence analyses". Bioinformatics, advance article online. doi: 10.1093/bioinformatics/bty395 - Read article - LaTeX preprint

FlexiDot versions and updates

Current version (29.06.2018): FlexiDot v1.04

For an overview of FlexiDot version updates please see the code history.

Older versions can be accessed in the code directory. Corresponding parameter cheat sheets are available as well.

Documentation

Implementation

FlexiDot is implemented in Python 2.7, using

Upon first starting FlexiDot, the program calls all needed modules. If absent, it installs them automatically using Python’s install manager pip. If this fails, please try again with administrator privileges.

Please note, that the dependency Biopython requires a C compiler. In case of errors during Biopython installation, installing Microsoft Visual C++ Compiler (Windows), GCC (Linux) or Apple’s XCode suite (Mac OS) may help.

Use FlexiDot

Download the FlexiDot script. With a right click on the field Raw you can download the script easily via Save as.

To run FlexiDot, Python 2.7 must be installed on the machine. FlexiDot is started via command line in the console. For a brief introduction to the command line interface, check out this nice tutorial.

In brief, the console can be started the following way:

  • Windows
    • start console: WINDOWS key + type CMD + ENTER (Shift + ENTER starts console as administrator)
    • prepare directory
      • select directory and add python script "flexidot.py" and sequence files
      • copy userpath from address bar (e.g.: C:\Users\Documents\Test)
    • navigate to directory in console: type cd userpath + ENTER (paste userpath using right click)
    • start Flexidot with the command below (with your specific fasta file name)
  • Linux/MacOS
    • start console: Applications → Utilities [Linux] or Accessories [MacOS] → Terminal
    • prepare directory (see above, e.g. /Users/Documents/Test)
    • navigate to directory in console: type cd userpath + ENTER (paste userpath using right click)
    • start Flexidot with the command below (with your specific fasta file name)

The general FlexiDot command depends on whether one or multiple fasta files are used as input via:

# use individual fasta file (can contain multiple sequences)
python flexidot.py -i input.fas [optional arguments]

# use multiple fasta files
python flexidot.py -i input1.fas,input2.fas [optional arguments]

# use all fasta files in current directory
python flexidot.py -a [optional arguments]

Optional arguments are explained below and in detail in the usage. Importantly, -k defines the word size (e.g. -k 10) and -t specifies the sequence type (-t y for DNA [default]; -t n for proteins). The plotting mode is chosen via -p and described below.

Plotting modes

FlexiDot allows sequence investigation in three run modes via the option -p/--plotting_mode:

-p 0 self sequence comparison -p 1 pairwise sequence comparison -p 2 all-to-all sequence comparison

Self dotplots

with -p/--plotting_mode 0

In self dotplot mode, each sequence is compared with itself. The resulting dotplots can be combined to form a collage [default] or written to separate files.

alt text

python flexidot.py -i test-seqs.fas -p 0 -D y -f 1 -k 10 -w y -r y -x n -m 6 -P 15 -g example.gff3 -G gff_color.config

Pairwise comparisons

with -p/--plotting_mode 1

For pairwise dotplots, the collage output is recommended for larger numbers of sequences. The collage output of the 15 pairwise dotplots for the test sequences is shown below. By default, dotplot images are in square format (panel A). This maximizes the visibility of matches, if the compared sequences differ drastically in length. To enable scaling according to the respective sequence lengths, the FlexiDot scaling feature is callable via option -L/--length_scaling (panel B). If scaling is enabled, a red line indicates the end of the shorter sequence in the collage output.

Panel A$ python flexidot.py -i test-seqs.fas -p 1 -D y -f 0 -k 10 -w y -r y -m 5 -c y -L n 
Panel B$ python flexidot.py -i test-seqs.fas -p 1 -D y -f 0 -k 10 -w y -r y -m 5 -c y -L y

All-against-all comparisons

with -p/--plotting_mode 2

In all-against-all mode, FlexiDot compares each pair from a set of input sequences. To enable the identification of long shared subsequences at a glance, FlexiDot offers similarity shading (switched on/off via option -x/--lcs_shading) based on the LCS length in all-against-all comparisons (see below).

python flexidot.py -i test-seqs.fas -p 2 -D y -f 0 -t y -k 10 -w y -r y -x y -y 0

Major features

Mismatch and ambiguity handling

In diverged or distantly related sequences matches may be interrupted by mismatches or residues might be represented as ambiguities to refer to frequent variants or mutations. Similarly, relaxed matching is helpful when analyzing error-prone sequences like SMRT reads. The achieved relaxation of the matching conditions thus increases sensitivity, while decreasing specificity.

Firstly, FlexiDot handles ambiguous residues, often found in consensus sequences. This allows the comparison of species-specific representations of multigene or repeat families as well as common variants or sequence subfamilies. The ambiguity handling is controlled via-w/--wobble_conversion Y/N.

Secondly, a defined number of mismatches within the window can be allowed with -S/--substitution_count [number of allowed mismatches (substitutions)]. This is even less stringent than the ambiguity handling. Please note, that only substitution mutations are allowed but not indels.

Lastly, both mismatch and ambiguity handling can be combined for the analysis.

Panel tl$ python flexidot.py -i Seq4.fas,Seq1.fas -p 1 -D n -f 0 -c n -k 10 -w n -r y -x n
Panel tm$ python flexidot.py -i Seq4.fas,Seq1.fas -p 1 -D n -f 0 -c n -k 10 -w n -r y -x n -S 1
Panel tr$ python flexidot.py -i Seq4.fas,Seq1.fas -p 1 -D n -f 0 -c n -k 10 -w n -r y -x n -S 2
Panel bl$ python flexidot.py -i Seq4.fas,Seq1.fas -p 1 -D n -f 0 -c n -k 10 -w y -r y -x n
Panel bm$ python flexidot.py -i Seq4.fas,Seq1.fas -p 1 -D n -f 0 -c n -k 10 -w y -r y -x n -S 1
Panel br$ python flexidot.py -i Seq4.fas,Seq1.fas -p 1 -D n -f 0 -c n -k 10 -w y -r y -x n -S 2

Annotation-based shading

Note: See also our tutorial on how to integrate annotation shadings with a real-life example.

In FlexiDot self dotplots, annotated sequence regions can be highlighted by shading to allow clear assignment of dotplot regions to specific sequence contexts (see Seq2 in self dotplots). The underlying annotation information must be provided in general feature format (gff3), either as individual file or file list via the -g/--input_gff_files option. To customize GFF-based shading, a user-defined configuration file can be provided via the -G/--gff_color_config option. Example files are provided in the test-data directory. Please note, that a legend is generated in a separate file.

If you wish to find out more on the gff3 file format used here, Ensembl provides a good overview.

python flexidot.py -i Seq2.fas -p 0 -D y -f 0 -k 10 -w y -r y -x n -m 12 -P 5 -g example.gff3 -G gff_color.config

[since FlexiDot_v1.03] Annotation-based shading also available for all-against-all dotplots

Previously only available for self dotplots, we added annotation-based shading to all-against-all dotplots, allowing for many new visualizations. As before, annotation information is provided as general feature file (GFF3). These features are added to the middle diagonal (see our example below).

Basic command:

python flexidot.py -i test-seqs.fas -g example2.gff3 -G gff_color.config -p 2

Command plus aesthetics as shown here (+ LCS shading, wordsize 10, change of subplot spacing and line width):

python flexidot.py -i test-seqs.fas -g example2.gff3 -G gff_color.config -p 2 -x y -k 10 -F 0.06 -A 1.5

The test files used here are provided:

Similarity shading

In all-against-all mode, FlexiDot compares each pair from a set of input sequences. To enable the identification of long shared subsequences at a glance, FlexiDot offers similarity shading (switched on/off via option -x/--lcs_shading) based on the LCS length (longest common subsequence, or longest match if mismatches are considered) in all-against-all comparisons. Longer matches are represented by darker background shading. A separate shading legend output file is created written according to mathematical interval notation, where interval boundaries are represented by a pair of numbers. Consequently, the symbols “(” or “)” represent exclusion, whereas “[” or “]” represent inclusion of the respective number.

FlexiDot similarity shading is highly customizable with the following parameters, explained in depth in the documentation:

  • Reference for shading (option -y/--lcs_shading_ref)
  • Number of shading intervals (option -X/--lcs_shading_num)
  • Shading based on sequence orientation (option -z/--lcs_shading_ori)

Shading examples based on sequence orientation (forward, panel A; reverse, panel B; both, panel C) are shown:

alt text

Panel A$ python flexidot.py -i test-seqs.fas -p 2 -D y -f 0 -t y -k 10 -w n -r y -x y -y 0 -z 0
Panel B$ python flexidot.py -i test-seqs.fas -p 2 -D y -f 0 -t y -k 10 -w n -r y -x y -y 0 -z 1
Panel C$ python flexidot.py -i test-seqs.fas -p 2 -D y -f 0 -t y -k 10 -w n -r y -x y -y 0 -z 2

Custom matrix shading

When comparing related sequences, multiple sequence alignments are frequently applied. The resulting pairwise sequence similarities can be integrated in the FlexiDot images by providing a matrix file via -u/--input_user_matrix_file <matrix.txt>. This allows a shading of the upper right triangle according to the matrix (here orange). With -U/--user_matrix_print y the matrix values can be printed into the respective fields. Besides, also text information can be provided in the matrix, but then shading is suppressed.

In the example, LCS and matrix shading are combined to visualize the relationships between different members of a repeat family.

python flexidot.py -i Beetle.fas -p 2 -x y -k 10 -S 1 -r n -u custom_matrix.txt -U y