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Welcome to the GeneParliamentID wiki! This page offers a broad overview of the GeneParliamentID (GPID) pipeline. For details on the following topics, please visit the dedicated wiki pages:

• Setup
• Pipeline inputs
• Building a reference dataset
• Method calibration
• Interpretation of results
• Tutorial

Overview

GPID is a pipeline for multi-gene DNA barcoding.

Starting from a set of multipe genes for a sample of unknown identity, the pipeline implements several filtering and calibration steps to provide a species-level identification. The identification explictly takes conflicting identifications between different genes into account and is combined with a confidence estimate of the identification.

For optimal results, method calibration using the provided companion script and test samples of known identity is recommended.

Inputs

• A set of multipe genes for a sample of unknown identity
• Reference data set containing the same genes for the lineage of interest
• Optional: calibration files to optimise pipeline performance

Key pipeline steps

1. Select genes with performance of species identification above user-defined threshold
2. Align each gene against reference, select top match based on highest Bit-score
3. Remove low-confidence matches that don't meet the user-defined alignment filtering thresholds
4. Summarise all remaining identifications to the Gene Parliament
5. Flag the sample as data-deficient if the number of genes (parliament size) is below the user-defined threshold
6. Select identification with most support as the top identification
7. Evaluate confidence in the top identification based on the percentage of genes supporting this identification

Outputs

The pipeline produces three outputs Example outputs of a test sample of a tropical mahogany timber sample.

Table of the Gene Parliament

Rank	Identification	Species Group	Support (%)	Support (count)
1	Entandrophragma angolense	Entandrophragma	75.82	138
2	Entandrophragma excelsum	Entandrophragma	16.48	30
3	Entandrophragma delevoyi	Entandrophragma	3.85	7
4	Entandrophragma congoense	Entandrophragma	2.75	5
5	Khaya ivorensis	Khaya	0.55	1
5	Swietenia humilis	Swietenia	0.55	1

Visualisation of the Gene Parliament

Table of top identification

Includes data check and confidence estimate. The data check is a test whether the user-definied minimum parliament size is met. The confidence estimate is based on the support for the top identification and method calibration using a set of test samples.

• Probability correct: correct identification to species
• Probability close: correct identification to user-defined species group
• Probability wrong: incorrect identification

Rank	Identification	Species Group	Support (%)	Support (count)	Parliament size (count)	Data check	Probabilty correct (%)	Probability close (%)	Probability wrong (%)
1	Entandrophragma angolense	Entandrophragma	75.82	138	182	PASS	100	0	0

Method calibration

To identify the optimal pipeline parameters for a given lineage, we strongly recommend conducting method calibration using the provided script. Method calibration also requires a set of test samples of known identity. The calibration script helps you to select the optimal parameters that balance accuracy of identification against retrievability of samples.

Method calibration includes the following steps:

1. Select the optimal alignment filtering thresholds
2. Calculate gene performance (percentage of correct identifications) following application of optimal alignment filters
3. Select optimal gene performance thresholds
4. Select threshold for minimum parliament size
5. Estimate confidence of identification depending on the percentage of genes supporting the top identification