Generation of MS-AFLP binary data with Beckmann CEQ8000

  1. Choose [Fragments] in main menu
  2. New Study > raw Data
  3. Select Analyis Parameter Set
    • MS-AFLP 600 bp > [Next] > [Analyze]
  4. Identify good tracks in Results list:
    • select all items > click right > Stacked Graphs
    • find bad tracks > click left on track to choose > click right and choose "Hide"
    • write name of these bad tracks down on paper
    • close window
  5. Generate MS-AFLP band call:
    • Remove bad tracks from study: in results list click left on sample > click right > choose "Remove from Study"
    • select all samples in the results list > click right > Stacked Graphs
    • Edit band calls:
      • change Y for each track so that size marker has same height in window
      • inspect peaks, click left to choose, click right for include (yes/no)
    • when finished click on symbol for Study Explorer to open Explorer
    • click left on [AFLP]
    • click right and chosse [New AFLP]
    • choose only D4 dye > run analysis
    • choose all bands and binary (1/0) format
    • export as CSV

Parsimony analysis

  • Use Excel to transpose data table (sample names in rows, band presence in colons)
  • use text editor to replace [tab] with nothing, count samples and count bands, generate PHYLIP-Pars input style
  • save in follwing format (found by trial and error):
    [10 spaces]number of individuals[12 spaces]number of 01
    [individual name in 7 letters][3 spaces]0101010101...[CR]
  • use PHYLIP-Pars (http://bioweb2.pasteur.fr/phylogeny/intro-en.html) for parsimony analysis
  • use PHYLIP-Drawgram to draw cladogram

Distance-Matrix analysis with Neighbour-Joining tree

  • create file format as above but with virtual restriction site size of 16:
    [10 spaces]number of individuals[12 spaces]number of 01[12 spaces]16
    [individual name in 7 letters][3 spaces]0101010101...[CR]
  • install Phylip locally and use RESTDIST
  • on message:
    restdist: can't find input data file "infile"
    Please enter a new file name>
    provide path to input file. NOTE! There is an unknown character length limit on the path+filename, keep it short and avoid SPACE and non-alphanumerica characters like -/ etc.
  • Modify parameter "site length":

    Restriction site or fragment distances, version 3.67

    Settings for this run:
    R Restriction sites or fragments? Sites
    N Original or modified Nei/Li model? Modified
    G Gamma distribution of rates among sites? No
    T Transition/transversion ratio? 2.000000
    S Site length? 6.0
    L Form of distance matrix? Square
    M Analyze multiple data sets? No
    I Input sequences interleaved? Yes
    0 Terminal type (IBM PC, ANSI, none)? ANSI
    1 Print out the data at start of run? No
    2 Print indications of progress of run? Yes

    Y to accept these or type the letter for one to change
    s
    New Sitelength?
    16
  • Modify parameter "Form of distance matrix":

    Restriction site or fragment distances, version 3.67

    Settings for this run:
    R Restriction sites or fragments? Sites
    N Original or modified Nei/Li model? Modified
    G Gamma distribution of rates among sites? No
    T Transition/transversion ratio? 2.000000
    S Site length? 16.0
    L Form of distance matrix? Square
    M Analyze multiple data sets? No
    I Input sequences interleaved? Yes
    0 Terminal type (IBM PC, ANSI, none)? ANSI
    1 Print out the data at start of run? No
    2 Print indications of progress of run? Yes

    Y to accept these or type the letter for one to change
    L
  • Start program:

    Restriction site or fragment distances, version 3.67

    Settings for this run:
    R Restriction sites or fragments? Sites
    N Original or modified Nei/Li model? Modified
    G Gamma distribution of rates among sites? No
    T Transition/transversion ratio? 2.000000
    S Site length? 16.0
    L Form of distance matrix? Lower-triangular
    M Analyze multiple data sets? No
    I Input sequences interleaved? Yes
    0 Terminal type (IBM PC, ANSI, none)? ANSI
    1 Print out the data at start of run? No
    2 Print indications of progress of run? Yes

    Y to accept these or type the letter for one to change
    Y
  • save "outfile" under new name (e.g. "restdistoutfile"), and change name "outfile" to "infile" in the PHYLIP folder
  • start program NEIGHBOR
  • change parameter L:

    Neighbor-Joining/UPGMA method version 3.67

    Settings for this run:
    N Neighbor-joining or UPGMA tree? Neighbor-joining
    O Outgroup root? No, use as outgroup species 1
    L Lower-triangular data matrix? No
    R Upper-triangular data matrix? No
    S Subreplicates? No
    J Randomize input order of species? No. Use input order
    M Analyze multiple data sets? No
    0 Terminal type (IBM PC, ANSI, none)? ANSI
    1 Print out the data at start of run No
    2 Print indications of progress of run Yes
    3 Print out tree Yes
    4 Write out trees onto tree file? Yes


    Y to accept these or type the letter for one to change
    L
  • type Y
  • save "outfile" and "outtree" under new name
  • vizualize "outtree" with TreeView

Cluster analysis for population structure analysis

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