T-RFPred, Terminal-Restriction Fragment Prediction
is a group of Perl scripts that will help researchers to identify the profiile peaks of a TRFLP fingerprint using clone libraries of partially sequenced 16S rRNA genes from the same sample. In silico methods, similar to this, are necessary to identify restriction fragment peaks. Basically this method estimates the expected size of your clone sequences for a given restriction enzyme. T-RFPred uses the aligned Genbank version of the Ribosomal DataBase Project (RDP) that will be reformatted to speed up the process. The idea behind this method is that in most circumstances, one can only have partial sequences in a clone library and not the whole 16S rRNA gene. Also, the primer that is used for the T-RFLP is usually the same as the one for sequencing and then about 20 bases at the beginning of the clone will be unknown.The method estimates the length of the fragment by adding up the length of the oligonucleotide, the estimate of the length of the "gap" and the length of the sample sequence from the beginning of the sample sequence up to the first target size of the restriction enzyme. The length of the "gap", missing bases between the end of the oligonucleotide and the beginning of the sample sequence, is estimated based on a number of sequences that are most closely related to the sample sequence in a subset of the Ribosomal Database Project sequences that are long enough to span the oligonucleotide. A local BLASTN finds the best hits and then Smith-Waterman alignments of the sample sequence and the best hits give accurate percent similarities. Also T-RFPred gives the taxonomy of the closest relative.
is a group of Perl scripts that will help researchers to identify the profiile peaks of a TRFLP fingerprint using clone libraries of partially sequenced 16S rRNA genes from the same sample. In silico methods, similar to this, are necessary to identify restriction fragment peaks. Basically this method estimates the expected size of your clone sequences for a given restriction enzyme. T-RFPred uses the aligned Genbank version of the Ribosomal DataBase Project (RDP) that will be reformatted to speed up the process. The idea behind this method is that in most circumstances, one can only have partial sequences in a clone library and not the whole 16S rRNA gene. Also, the primer that is used for the T-RFLP is usually the same as the one for sequencing and then about 20 bases at the beginning of the clone will be unknown.The method estimates the length of the fragment by adding up the length of the oligonucleotide, the estimate of the length of the "gap" and the length of the sample sequence from the beginning of the sample sequence up to the first target size of the restriction enzyme. The length of the "gap", missing bases between the end of the oligonucleotide and the beginning of the sample sequence, is estimated based on a number of sequences that are most closely related to the sample sequence in a subset of the Ribosomal Database Project sequences that are long enough to span the oligonucleotide. A local BLASTN finds the best hits and then Smith-Waterman alignments of the sample sequence and the best hits give accurate percent similarities. Also T-RFPred gives the taxonomy of the closest relative.If you are interested in our method and want to know more follow this link or click on the METHOD tab.