Tutorial

It is a tutorial on how to analyze genome approximate repeats by MUMMER and our tool ApproxRepeatAnalyzer.

Suggested operating system : Mac OS X / Linux

Analysis of exact and approximate repeats by MUMMER

Here is a step-by-step tutorial to download, run and experiment with MUMMER.

1. Open up your terminal.

2. Download and Unpack MUMMER from,

    http://sourceforge.net/projects/mummer/files/mummer/3.23/
   

3. Change to the directory that contain MUMMER and build it by,

    make install
   

4. Download a test genome (Ecoli 536 in this example) from NCBI by,

    perl -e 'use LWP::Simple;getstore("ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Bacteria/Escherichia_coli_536_uid16235/CP000247.fna","ecoli.fasta");'
   

5. Extract exact repeat statistics with exact repeat length >= 20,

    ./mummer -maxmatch ecoli.fasta ecoli.fasta > outputFileKK
   

6. View the MUMMER output of the exact repeats.

    more outputFileKK
   

7. You will see something like the following. The first column is the startlocation of repeat copy 1 , second column is the start location of repeat copy 2 and the third column is the length of the exact repeat.

   gi|110341805|gb|CP000247.1|

   1 1 4938920

   143740 9820 51

   646218 9822 49

   ...

8. We need to create an alignment file.

    ./nucmer --maxmatch --nosimplify ecoli.fasta ecoli.fasta
   

   The output will be in a file called out.delta

9. We can then obtain approximate repeat statistics with repeat homology of >= 95% with length >= 500 by,

    ./delta-filter -l500 -i95 out.delta > filter.delta 
   

10. Let us look at the filter.delta

     more filter.delta
    

    and you will see something like

    gi|110341805|gb|CP000247.1| gi|110341805|gb|CP000247.1| 4938920 4938920

    1 4938920 1 4938920 0 0 0

    0

    227625 232949 4418733 4424057 12 12 0

    0

    227694 232953 3538642 3533381 8 8 0

    -6

    -2801

    0

    227785 229525 4241245 4242985 6 6 0

    -44

    1690

    0

11. Here is the official explanation of what these numbers mean (by the MUMMER manual http://mummer.sourceforge.net/manual/)

          Following this sequence header is the alignment data. 
     Each alignment following also has a header that describes 
     the coordinates of the alignment and some error information. 
     These coordinates are inclusive and reference the forward 
     strand of the DNA sequence, regardless of the alignment
     type (DNA or amino acid). Thus, if the start coordinate is 
     greater than the end coordinate, the alignment is on the reverse 
     strand. The four coordinates are the start and end in the 
     reference and the start and end in the query respectively. The
     three digits following the location coordinates are the number 
     of errors (non-identities + indels), similarity errors 
     (non-positive match scores), and stop codons (does not apply to
     DNA alignments, will be "0"). An example header might look like:

       2631 3401 2464 3234 15 15 2
       Notice that the start coordinate points to the first base in the first codon, 
     and the end coordinate points to the last base in the last codon. 
     Therefore making (end - start + 1) % 3 = 0. This makes
     determining the frame of the amino acid alignment a simple 
     matter of determining the reading frame of the start coordinate 
     for the reference and query. Obviously, these calculations are 
     not necessary when dealing with vanilla DNA alignments.
       
       Each of these alignment headers is followed by a string of 
     signed digits, one per line, with the final line before the next
     header equaling 0 (zero). Each digit represents the distance 
     to the next insertion in the reference (positive int) or deletion 
     in the reference (negative int), as measured in DNA bases OR
     amino acids depending on the alignment data type. For example
     , with the PROMER data type, the delta sequence (1, -3, 4, 0) would 
     represent an insertion at positions 1 and 7 in the translated 
     reference sequence and an insertion at position 3 in the
     translated query sequence. Or with letters:
     
     
     A = ABCDACBDCAC$
     B = BCCDACDCAC$
     Delta = (1, -3, 4, 0)
     A = ABC.DACBDCAC$
     B = .BCCDAC.DCAC$
  

11. You may now want to explore vaious functions offer by MUMmer by looking at their manual(pointer above). Some recommended and useful commands include show-coords and mummerplot.

Indepth analysis of the approximate repeat with our ApproxRepeatAnalyzer pipelined with MUMMER

Here is a step-by-step commands to pipeline MUMMER with our approximate repeat analyzer (ApproxRepeatAnalyzer)

1. Download the files in this repository.

2. Generate length-10 sliding window plot to view the neighborhood and interior of an approximate repeat by

    python repeatStat/slidePlot.py "ecoli.fasta" "setup" 227625 4418733 10000 2000
   

   This commands means to plot the repeat of ecoli.fasta with repeat copies starting location being 227625, 4418733. The plot will span 10000 base pairs within/beyond the approximate repeat. We show 2000 base pairs before the starting locations.

   An example plot is shown here.

3. You can run the following to generate a couple of plots to understand the approximate repeats.

    python repeatStat/repeatStatMain.py "~/Downloads/MUMmer3.23/mummer" "ecoli.fasta" "Escherichia_coli_536" 30
   

   Here "~/Downloads/MUMmer3.23/mummer" is where you put MUMMER, "ecoli.fasta" is the genome file, "Escherichia_coli_536" is the working folder, and 30 is the number of long seed exact repeats to be used to generate the plots.

4. Let us understand what these plots tell us here. (Hamming and edit distance are both analyzed, but for simplicity, let us focus on Hamming distance here)

   a) Extension plot to compare the neighborhood and interior of several long approximate repeats. It measures how the repeats are extended beyond its longest duplicating segment. A big jump suggest that the repeat has some sparse SNPs while the steady growth beyond certain point suggest that it reaches the random flanking region. This graph is plotted when we extend to both sides of a repeat. This graph is plotted when we combine the extension from both sides, by greedily using the one that have larger extension to come first.

   b) Classification of approximate repeats and its spectrum i) Scatter plot to characterize the extension and SNP rate of the long approximate repeats.

   This is shown in the top graph. For each approximate repeat, we plotted it charateristics on this 2D plot, with its color code being the length of teh approximate repeats.

   ii) Combined repeat spectrum of simple, interleaved and triple exact/approximate repeats.

   This is shown in the bottom graph. The red spectrum is for exact repeats while the blue spectrum is for approximate repeats. The green line is the longest exact repeat.

   c) Interleaved and triple approximate repeats

   Moreover, we have the files that show the spectrum of interleaved and triple approximate repeats and their corresponding scatter behaviour. ..