ProbeSpec is implemented in ARB’s PROBE_DESIGN class utilizing its prefix tree database server. ProbeSpec’s functionality is implemented in the classes ArbProbe and ArbProbeCollection (abstraction of OP sequences and import/export functionality), ArbProbeMatchWeighting (providing weighting matrices for position specific nucleotide substitutions), ArbMatchResult, ArbMatchResultSets and ArbMatchResultsManager (abstraction of OP to PT-Server sequences with given weighting matrices and maximum number of mismatches) and ArbStringCache (providing string to disk caching of match string results).

Probe specificity calculations in ProbeSpec are based on the initial mismatch penalties given by a 4×4 substitution matrix for all possible nucleotide substitutions. Additionally, each mismatch penalty is weighted based on the position of a mismatch in the probe: mismatches at the ends of an OP are less likely to affect the binding of complementary sequences than mismatches in the center of a probe. Positional weights are calculated as follows: for a mismatch at position p in a given OP sequence of length l a weight W is calculated with

W = e S P 2 ( 1 )

where

S = − In ( 10 ) w ( 2 )

and

P = 2 * p − l l − b ( 3 )

The weight distribution given by (1) follows a bell curve penalizing mismatches at either end of the OP sequence less than mismatches in the center of the sequence. The user defined parameter w in equation (2) controls the spread of the weight distribution; user defined parameter b in equation (3) controls the midpoint and therefore enables the user to increase positional weights on either side of the OP sequence. For default parameters of w=1 and b=0, positional weights range from a minimum 0.1 for mismatches at the first and last nucleotide in the sequence to a maximum of 1 for mismatches at the center.

For user interaction with ProbeSpec ARB’s general user interface was extended with four new dialog windows: (i) a Probe Collection dialog, (ii) a Probe match with specificity, (iii) Match display control dialog and (iv) a Tree Marker settings dialog ( Figure 1).

The Probe match with specificity is the main entry point of ProbeSpec. It displays all loaded probes which can be edited, imported and exported through the Probe Collection dialog. Additionally, the Probe Collection Dialog allows the user to change the default settings for substitution penalties and positional weight parameters.

The main GUI of ARB was extended to graphically represent the probe matching results: each probe is represented by a colored vertical bar indicating a match of the OP to the specific phylogenetic group. Incomplete cover of a phylogenetic group is represented by transparency of a bar: the fewer members of a group that are covered by a given probe the higher the transparency of a bar is.

The dialogs Match Display Control and Tree marking settings enable interactive adjustment of probe match parameters such as mismatch threshold, group marked and group partially marked threshold.

ARB and the included ProbeSpec functionality can be run on any common PC, laptop or workstation. However, we recommend system specifications of at least 4GB of RAM and a dual-core processor to run ProbeSpec.

For any probe development, ProbeSpec requires a phylogeny of target sequences and organisms that the OPs should match to as well as a list of potential OPs.

For an introduction to sequence analysis using ARB, please refer to the main ARB documentation at http://www.arb-home.de/documentation.html. For evaluation purposes a sub-set of the data published in Krausfeldt et al. (2017) can be found on Zenodo ¹⁰ . To set up ARB select the provided nitrifyers_2017_04_for_paper.arb database file on start of ARB. To be able to run ProbeSpec a PTServer has to be created from the database via the Probes tab and the PT_Server Admin option in the PT Server Admin widget. Select the loaded database and click Build server. After completion close the progress bar and the PT Server Admin widget.

Before running a batch specificity test, a probe collection, i.e., a list of probes to be tested, has to be created using the Probe Collection window where probes can be added to and removed from a collection: Open the Probe Match with Specificity window via the Probes-tab in ARB ( Figure 1A) and select Edit ( Figure 1B) to open the Probe Collection window ( Figure 1C). To open the provided test data set use the load button and select the provided amoA70mers.xpc probe collection. Additionally, the sequence of new probes can be entered into the Target String text field. To add new OPs to the collection press Add. Probe collections can also be in this dialog.

The Probe Collection window can be used to define the specificity measures used by ProbeSpec to identify matching probes. This includes the definition of specific mismatch penalty values as well as the values for bias b and weight w (see subsection Operations in the Methods section for details).

After creation of a probe collection and configuration of the match parameters the Probe Collection window can be closed and the specificity search can be started by clicking the Match button ( Figure 1B). A status dialog will appear and show the progress of the search.

The final match results are shown in the ARBs main window: each matching probe is represented by a coloured bar next to the group/clade the probe matches with the given thresholds ( Figure 1A). The visualization can be configured using the two dialogs Match Display Control ( Figure 1B) and Tree Display settings ( Figure 1C), the latter of which can be accessed via the Marker Display Settings button on the Match Display Control widget.