MicrosatDesign is a pipeline tool for transforming
sequencer trace files into DNA markers

1. The Copyright of MicrosatDesign

Copyright ©2004 by Daphnia Genomics Consortium and Indiana University.
All Rights Reserved.

MicrosatDesign is a pipeline tool that (i) identifies microsatellites from DNA sequencer trace files, (ii) designs optimal primers for amplifying microsatellite markers and (iii) extracts the amplicon sequences. MicrosatDesign integrates a suite of computational programs that, when combined, automatically builds a microsatellite primer database from the raw output of genome sequencing projects. MicrosatDesign is freely distributed to the scientific community on the condition that it will not be used for commercial gain by any individual or organization. Any commercial use of MicrosatDesign, or any of its parts, is prohibited without written permission by its authors.

The authors and the DGC make no guarantee of any kind regarding its usefulness and disclaim all warranties, expressed or implied, without limitation.

2. How to reference MicrosatDesign

Although this program is within the public domain, please reference this work.

Singan, V.R. and J.K. Colbourne (2004) MicrosatDesign is a pipeline for transforming sequencer trace files into DNA markers. Available online via: http://daphnia.cgb.indiana.edu/wfleabase/software/

Click Here to Download Program

3. News - Version 2.0 is now available (February 22, 2005)

Version 2.0 is now available and contains substantial improvements over the previous release. In contrast to version 1.0, users can now specify the application of either Pipeline 1 using GCG Findpatterns or Pipeline 2 using EMBOSS etandem, depending on whether they have access to commercially available GCG software or to freely available EMBOSS programs.

4. Configuration and Execution Instructions

MicrosatDesign is a perl script that works on any UNIX based computer platform where Perl v5.8, Phred/Phrap, Primer3 and GCG FindPatterns or EMBOSS etandem are installed.

1. Download MicrosatDesign_v2.0.tar.gz into your working directory


2. Extract the program by executing at the command prompt: gunzip MicrosatDesign_v2.0.tar.gz


3. Then execute at the command prompt: tar -xvf MicrosatDesign_v2.0.tar


4. Please find within the newly created directory MicrosatDesign_v2.0 a "README" file, the program "MicrosatDesign.pl", the configuration file "MsD.conf", an "example" directory and a "lib" directory. If you care to modify the search patterns used by GCG FindPatterns, this can be done within the "pattern.dat" file found within the "lib" directory.


5. Modify the configuration file "MsD.conf" to specify the correct directories for all modules of the program by (a) setting MsDHOME to the installation directory of MicrosatDesign; (b) setting TRACEFILE to the directory containing sequencing trace files; (c) setting MOLBIO to the GCG installation directory; (d) setting GCGSTARTUP to the GCG startup script; (e) setting GCGINIT to the GCG initialization script; OR instead of c-e, (f) setting EMBOSSDIR to the EMBOSS installation directory, (g) setting PLPLOT_LIB to the directory containing PLPLOT libraries, (h) setting EMBOSS_ACDROOT to the directory containing all EMBOSS acd files, (i) setting EMBOSS_PATH to the EMBOSS executable path, (j) setting PERCENT_IDENT to the percentage identity for specifying imperfect dinucleotide repeats, (k) setting REPEAT_COUNT to the minimum number of repeats for imperfect dinucleotide repeats, AND (l) setting PHRED to the phredPhrap executable path; (m) setting PRIMER3 to primer3 executable path.


6. To use the program, type at the command prompt: perl MicrosatDesign.pl -program <gcg OR emboss>

If the <directory_of_trace_file> is named chromat_dir, the results are written to the same directory, else a new directory named chromat_dir is created at the same level as the <directory_of_trace_file> and results are written within it.

The four integrated programs phredPhrap, GCG FindPatterns, EMBOSS etandem and Primer3 are set to execute in default options. If tweaking of these options need be done, they can be done by modifying the calls to these programs in MicrosatDesign.pl

5. Interpretation of Results:

The results are written into two directories: chromat_dir and phd_dir.

phd_dir - is the directory created by phredPhrap and contains base calls and quality information of all the trace files.

chromat_dir - based on the user option of the program (GCG/EMBOSS), chromat_dir contains:

For GCG,

MicrosatDB.out, <base_dir_name>.fasta, <base_dir_name>.rsf, findpatterns.find, findpatterns.rsf, primer3.in, primer3.out

For EMBOSS,

MicrosatDB.out, <base_dir_name>.fasta, alltrace.tan, primer3.in, primer3.out

1. MicrosatDB.out - This file contains the resulting Amplicon IDs, matching primer pair sequences, their GC nucleotide compositions, predicted melting temperatures along with the descriptions of the microsatellite motifs and amplicon sequences.


2. <base_dir_name>.fasta - This file contains the multiple fasta format sequences of all the trace files.


3. <base_dir_name>.rsf - This file is the RSF (Rich Sequence Format) of the trace files created from the fasta file.


4. findpatterns.find - The output of GCG FindPatterns contains matches to microsatellite motifs.


5. findpatterns.rsf - This file is the RSF (Rich Sequence Format) of the GCG FindPatterns results.


6. alltrace.tan - This file containts concatenated output of EMBOSS etandem for all the sequences.


7. primer3.in - This file is the input to primer3 in BoulderIO format, created from findpatterns.rsf.


8. primer3.out - This file is the output of Primer3 containing primer pairs for amplifying microsatellite markers.

<base_dir_name> is the directory under which the chromat_dir is created.

6. Example Input and Output files

A sample tracefile directory with 16 trace files is provided in the example directory under both gcgexample and embossexample sub-sections. The output directories chromat_dir and phd_dir are also within each of the example directories.

The MicrosatDB.out in chromat_dir contains the amplicon database for the example tracefiles.

7. Reporting problems:

Please report problems and bugs within MicrosatDesign on the DGC Collaboration Wiki BugReports. https://daphnia.cgb.indiana.edu/

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