The RIFLE system compares restriction patterns of possibly unknown microorganisms against a database of theoretical restriction patterns generated from a 16S rDNA database, e.g. the RDP database. It returns a list of microorganisms whose theoretical restriction patterns match the laboratory patterns. The following scheme gives an overview of the complete method used.
The principle of the generation of restriction patterns in the laboratory is shown in phases (1) to (4). The experimental details will only be discussed here if they are important for the comprehension of the RIFLE parameters. For a detailed description of an experimental method yielding restricton patterns see [WAP95].
The method used to generate the theoretical restriction patterns is shown in phases (5) to (8).
RIFLE has been designed to behave very sensibly in the presence of
experimental errors as well as uncertainty in database entries.
Restriction patterns of multiple restriction enzymes can be combined to
improve the quality of identification, additional parameters allow the
individual adaptation to laboratory processes, e.g. exactness of fragment
length determination. The following section describes the parameters
of the RIFLE system.
Parameters of the RIFLE system
Databases
RIFLE compares your restriction patterns against theoretical
restriction patterns which are generated from 16S rDNA databases. As a
basis for pattern comparison, you may select one or more databases
from the list.
If more than one database is selected, the same
organism name may appear more than once in the results because it is
contained in more than one database.
If you would like to compare your restriction patterns against a database that is not listed, just indicate this database to the RIFLE Administration: bibi-help(at)techfak.uni-bielefeld.de.
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Primer
The selection of PCR-amplified sequences (Phase 6 in the scheme) simulates the
PCR-amplification of 16S rDNA sequences in the laboratory. For example,
the primer pair R1n-U2 amplifies a subsequence of 1063 base
pairs of the 16S rDNA of E.coli which starts at position 22
and ends at position 1085.
You have to select one of the indicated primer pairs. If you would
like to use a primer pair that is not yet contained in the
list, please indicate the desired primer pair to the
RIFLE Administration:
bibi-help(at)techfak.uni-bielefeld.de
Experimental artefacts
Some laboratory processes produce additional sequences before or after
the 16S rDNA primers, e.g.
vector sequences.
Please indicate the length of these sequences. The
RIFLE system will suppose
that these sequences are not cut by the selected restriction
enzymes. Their length will be added to
the length of the first/last fragment of the 16S rDNA.
A length of zero means that no experimental artefacts have been produced.
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Restriction Patterns
The restriction patterns generated in the laboratory can be entered as
a sorted or unsorted list of fragment lengths. The numbers have to be
seperated by commas or blanks. For each pattern, a pattern name should
be indicated. For each group of restriction patterns, the enzyme which
has been used for the digestion has to be selected in the list of
restriction enzymes. If you would like to use enzymes which are not
yet contained in the list,
please indicate the desired enzymes to the
RIFLE Administration:
bibi-help(at)techfak.uni-bielefeld.de
To improve the quality of identifications, the results of multiple digests of one strain with different enzymes can be combined. If you activate the button "Restriction patterns with the same number originate from the same strain", restriction patterns with the same second number, e.g. 0.2 and 1.2, will be considered as different digests of the same strain, the results will be combined. The combined results will appear in the "Combined results" section of the RIFLE response.
Tests have shown that 3-4 enzymes per strain yield reliable identifications.
Due to poor database sequence quality, restrictions pattern data may be missing for some sequence-enzyme combinations. If you activate the button " Organisms for which restriction pattern data is missing...", these organisms will not be listed in the "Combined results" section.
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Laboratory resolution
Restriction fragment length determination is normally performed by gel
electrophoresis and subsequent silver staining. Unfortunately, the
exactness of this method is limited. Short fragments are often not
detected, and two fragments with the same or very similar length may
appear as one single band in the gel. RIFLE can take these limits
of laboratory resolution into account by adapting the theoretical
restriction patterns prior to comparing them to the lab patterns.
If the value of the field "Fragments shorter than..." is non-nil, theoretical fragments shorter than the indicated value will be deleted prior to pattern comparison.
If the value of the field "Subsequent fragments..." in non-nil, subsequent theoretical fragments whose length difference is smaller than the indicated percentage will be considered as one fragment. This reflects the clustering of several bands into one single band that may appear in the gel. If the percentage is set to zero, patterns are compaired "as they are".
Both options are only activated if the number of fragments in the theoretical pattern and the laboratory pattern differ.
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Filtering of results
To reduce the length of the identification lists generated by RIFLE,
you may limit the number of organisms which are contained in the
lists. You may indicate the maximum number of organisms displayed as
well as the maximum edit distance between the theoretical restriction
patterns and your laboratory restriction patterns.
If restriction patterns of unknown organisms are generated by using only one restriction enzyme, random similarities between theoretical restriction patterns and the laboratory patterns may lead to false identifications. Therefore the use of several enzymes on the same sample is recommended. Test with the RIFLE system have shown that three to four restriction enzymes per sample are a good compromise between laboratory costs and reliable identifications.
Obviously, organisms can only be correctly identified if their theoretical restriction patterns are contained in the RIFLE restriction pattern databases. Therefore, for each database used, the RIFLE results contain a link to a list of organisms for which theoretical restriction patterns exist.
The example results show the identification of a strain named L8. For this strain restriction patterns as well as the complete 16S rDNA sequence amplified with the primer pairs R1n-U2 have been determined. When using direct sequence comparison, the marine snow associated str. agg8 showed the highest similarity to L8.
The independent results for the restriction patterns generated with the enzymes HpaII and HinfI show str. agg8 on the second position in the identification list. The combined results show str. agg8 on top of the identification list. So the combination of the two enzymes yields the same result as the identification by direct sequence comparison, the unknown strain L8 has been correctly identified.
Any comments, suggestions or technical problems? Please send email to the bibi-help(at)techfak.uni-bielefeld.de
Author:Henning Hermjakob, ihhermja@techfak.uni-bielefeld.de
Last modified $Date: 2002/10/28 10:48:41 $ by $Author: hmersch $.
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