BEsTRF: a tool for optimal resolution of terminal restriction fragment length polymorphism analysis based on user defined primer-enzyme-sequence databases
Supplementary material

Reference for citation:

BEsTRF: a tool for optimal resolution of terminal-restriction fragment length polymorphism (T-RFLP) analysis based on user defined primer-enzyme-sequence databases
Blaz Stres; James M. Tiedje; Bostjan Murovec
Bioinformatics 2009 25(12):1556-1558; doi:10.1093/bioinformatics/btp254

Free access link to abstract:


Blaz Stres, University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Chair for Microbiology and Microbial Biotechnology, Groblje 3, 1234 Domzale, Slovenia

James M. Tiedje, Michigan State University, Department of Soil Science and Center for Microbial Ecology, Plant and Soil Science Building 540, MI-48828 East Lansing, USA

Bostjan Murovec, University of Ljubljana, Faculty of electrical engineering, Laboratory for Computer Integrated Manufacturing, Trzaska cesta 25, 1000 Ljubljana, Slovenia


The current protocol describes a suitable procedure for using BEsTRF local tool for determining the optimal configuration of T-RFLP. BEsTRF provides an in-depth and controlled environment for an up to date exploration of Primers-Enzymes-Gene sections combinations used in T-RFLP, simulating PCR, fragment generation and analysis. User defined sequence database can be processed and the resolution of user specified sets of primers and restriction endonucleases can be analyzed on either forward or reverse terminal fragments thus exploring the vast multidimensional space of forward and reverse primers, restriction endonuclease and sequence detection specificity. In addition, BEsTRF can be used to generate "virtual clone libraries", sequence subsamples, retrieved based on primer specificity for downstream phylogenetic analyses.

Click to enlarge

BEsTRF in action

Basic information regarding T-RFLP can be obtained at

BEsTRF Binaries are available for 32-bit Microsoft Windows® as well as for 32-bit Linux® platforms.

BEsTRF Version 3.6: Microsoft Windows®, Linux®.

BEsTRF Version 3.5: Microsoft Windows®, Linux®.

BEsTRF Version 3.4: internal release only.

BEsTRF Version 3.3: Microsoft Windows®, Linux®.

BEsTRF Versions prior to 3.3: internal releases only.

The latest version 3.6 dates from 2009_May_19. Changes from version 3.5 are as follows:
  1. (Minor) File "screen_output.txt" is renamed to "progress_report.txt".

  2. (Minor) Corrected missing warning about duplicate Primers and Enzymes in file "progress_report.txt" (previously "screen_output.txt").

  3. (New feature) BEsTRF now generates new T-RF Phylogeny report. Please examine new sections 34 and 35 of file "BEsTRF_params.txt" for details.

Version 3.5 dates from 2009_April_30. Changes from version 3.3 (the first publicly available version from 2009_March_20) aim to improve users' experience with the program but otherwise do not introduce any modifications of the underlying operation. The Summary of changes is as follows:
  1. Screen output has been vastly reduced out of necessity since version 3.3 generated significantly more text than a user could comprehend.

  2. BEsTRF now assists better in documenting archived analyses by storing parameters and other information about finished work in a subdirectory "log_of_analysis", which is created under main directory with results of an analysis.

  3. Previous comprehend screen output is now written to file "screen_output.txt", which is stored in directory "log_of_analysis". A user can now easily examine this file after the analysis to spot any potential problems. Before, the same examination required unfeasibly fast reading of console output and was therefore not user friendly.

  4. A summary of parameters is now stored in directory "log_of_analysis" in two different ways, for which two new files "effective_params_1.txt" and "effective_params_2.txt" are generated. Both of them contain the same information, albeit organized in different ways. You may utilize whichever one suits your preferences.

  5. The utilized parameters file is now copied unchanged into directory "log_of_analysis". This way potential user's comments in the file are also archived along other data about an analysis.

  6. Directory "log_of_analysis" now contains file "Version_and_date.txt", which documents starting time of analysis and utilized BEsTRF version.

Please note: BEsTRF manual does not yet reflect these changes, but the document is nonetheless still fully relevant.

The utility is console oriented. All parameter are listed in the so-called parameters file, which the user specifies upon invocation of the program, either as the BEsTRF parameter, like this:

BEsTRF file_with_parameters.txt

Alternatively, BEsTRF may be invoked without any parameters, in which case the program asks for the parameters file name at a console prompt.

Click to enlarge

BEsTRF route of an analysis

The usage of BEsTRF is comprehensively described in the BEsTRF manual (currently for version 3.3, but new features are utilized easily without detailed instructions).

A comprehensive example and a short summary of parameters file are also available, as well as a README file.

Worked Examples

Several worked examples (produced with BEsTRF version 3.5) of various genes explored so far were prepared based on aligned sequence collections of Functional Gene Database / Pipeline ( that are available here for download and execution at your local computer as ready-to-run ZIPped files:

A-CoA-analysis (ZIP 4.5 MB).
bphA-analysis (ZIP 12.4 MB).
nirK-analysis (ZIP 12.8 MB).
nirS-analysis (ZIP 91.3 MB).
cnorB-analysis (ZIP 4.3 MB).
qnorB-analysis (ZIP 3.0 MB).
nosZ-analysis (ZIP 34.9 MB).
nrfA-analysis (ZIP 4.5 MB).
rpoB-analysis (ZIP 141.6 MB).
tetW-analysis (ZIP 26.5 MB).

archaea-analysis (0.3 MB; without sequences database and precomputed results files). Please, see the accompanying README_first.txt file.
bacteria-analysis (0.3 MB; without sequences database and precomputed results files). Please, see the accompanying README_first.txt file.

All 10+2 examples include preset BEsTRF parameters' files for performing ten distinct analyses (ten within a scope of each example) and Windows executables together with batch files for launching of the prepared analyses. Linux executables can be downloaded manually from the above link.

Thus there are ten distinct BEsTRF parameter files for each example at hand. These parameters differ in the number of allowed primer mismatches, insertions and deletions as well as recognition of degenerated primer binding sites.

As an alternative to downloading of examples, you can browse directory structure with unpacked examples together with results (except for anarchaea and bacteria examples). However, note that you cannot run BEsTRF program without downloading it first.

Please note that the analyses of the archaeal and bacterial 16S rRNA genes are provided here without underlying sequence databases and results due to their size (multi 40 Gb sets). The former can be obtained as compressed files at:

*RESOURCES of the Ribosomal Database Project II (;jsessionid=81D78ABD098F7AF5EA2CC102FFAC0F4C)
*DOWNLOAD of the Greengenes (
*DOWNLOAD of the ARB - silva (

Aligned sequence collections will decompress to 2-30 GB in size.

License information

The material in this page may be freely used for academic, educational and any other non-profit usage.

Please cite the above reference if you find this material useful.


The material in this page is developed and disseminated with the best effort to make it error-free and working as described. However, there are no guarantees.

We, the developers do not take any responsibilities whatsoever if the use or misuse of the material causes or leads to personal injuries, data loss, material damage or any other undesired consequences.

Under no conditions should the developers be charged or prosecuted by anyone using the program for any reason whatsoever.

Please report bugs and wishes to The email is also intended for providing help and feedback, as well as receiving questions and comments regarding BEsTRF.

Two the most missing features: graphical user interface and online processing, are already foreseen.