Sequence variants are amino acid substitutions that occur along the primary structure of a protein, either by genetic mutation of a clone’s DNA or by misincorporation during cell culture. It is often an arduous task taking hours or sometimes days to distinguish true and false positives from the search results.
Many analysts who analyze sequence variants often do so by implementing a result filtering strategy based on empirically-derived criteria to reduce analysis time. Byos comes already equipped with various validator algorithms which assist analysis or filter for classes of modifications such as sequence variants. The algorithms can be set during project creation and the results displayed next to the identification. Having the validation occur with the search result so that the analyst can quickly inspect the result of the filter further speeds up analysis because time is not spent going back and forth between an external filtered list and the search result.
Activating the Byos SVA Validator
Add the following text in Byos in the Advanced Commands section during project creation to activate the SVA Validator algorithm and to run it with the default parameter values.
SvaEnabled = true
The validator will then label sequence variants in the Comments column in Byos as Critical, Warning, or Clear to describe the severity as well as a description of the reason for misidentification. If the default parameter values are not sufficient, they can be altered by adding the following text to the Advanced Commands and modifying the parameters accordingly. Note that the default parameter values are displayed here.
DeltaModScoreThreshold = 10;
ScoreThreshold = 225.0;
PercentXICRatioThreshold = 1.0;
FwhmThreshold = -1;
PredictedTimeDiffThreshold = 5.0;
PPMStandardDeviations = 3;
CommonModLookBothWaysFromModResidue = 3;
SVA Validator Labeling Logic
For a complete explanation of the parameters, please refer to the Peptide Workflows manual and the Release notes for recent releases.
SVA Validator Flags and Their Use
The flags in the Comment column can then be used to quickly ascertain whether an identification is a true or false positive. Typically, anything the validator labels as Critical is very likely a false positive and only a brief confirmatory review is required. Identifications labeled as Warning should be manually inspected. Having the reason for the warning will allow an analyst to quickly check the criteria used. Any identification labeled as Clear has passed all the filters and is likely a true positive. In the example below, the selected ID is labeled Clear: No Flags. The shift in y-series at the appropriate residue strongly suggests that this is indeed a sequence variant.
The comments can also be used for quickly filtering true positives and generating reports, though manual inspection is still highly encouraged.
Example of report using filtering on comment flags.
Validators in Byos can be used to quickly process search results by assisting in the data validation of peptide identifications. Though the topic here is specifically sequence variants, Byos also provides functionality for validating common modifications, e.g. oxidation, deamidation, etc., as well as another validator for disulfide bond analysis.