MixDeR - Current Version: 0.7.1

MixDeR (Mixture Deconvolution in R) is a workflow (with a Shiny app) for performing mixture deconvolution of ForenSeq Kintelligence SNP data for two-person mixtures using EuroForMix and creating GEDmatch PRO reports for the individual contributor SNP profiles.

This method requires extensive validation of the settings. MixDeR provides the option of calculating various metrics for evaluating the accuracy of the deduced SNP genotypes. This is extremely useful when determining settings, specifically the allele 1 probability threshold, the allele 2 probability threshold, and the minimum number of SNPs.

Note: MixDeR (and EFM) assume the mixture samples are composed of two contributors. MixDeR is able to identify and alert the user to samples that may be potentially either single source or consist of a mixture with a large mixture ratio (i.e. > 1:100 ratio between contributors). In these scenarios, the user is warned to be cautious with the minor contributor inferred genotyping results.

The preprint of the manuscript on MixDeR is available here.

Installation

For any installation, EuroForMix must be installed. Please follow the instructions from the EuroForMix GitHub page. For this version of MixDeR, EFM version 4.0.8 or earlier is required.

If installing from GitHub:
The R package devtools is required to install from GitHub:

install.packages("devtools")
devtools::install_github("bioforensics/mixder")

If installing from source, first install the following R packages:

install.packages(c("dplyr", "ggplot2", "glue", "prompter", "readxl", "rlang", "shiny", "shinyFiles", "shinyjs", "tibble", "tidyr"))

To install MixDeR from source (i.e. the mixder_0.1.0.tar.gz file):

install.packages("/path/to/mixder_0.1.0.tar.gz", repos = NULL, type="source")

For example, if the mixder_0.1.0.tar.gz file is located in your Documents folder:

install.packages("~/Documents/mixder_0.1.0.tar.gz", repos = NULL, type="source")

Usage

To launch the shiny app:

library(mixder)
mixder()

Required files

1. Mixture Kintelligence SNP profiles
   This can be in the form of the UAS Sample Report or a TSV file with the below format:

Sample.Name	Marker	Allele.1	Allele.2	Height.1	Height.2
Sample01	rs12615742	T	C	0	134
Sample01	rs16885694	G	A	43	63

The files should be tab delimited and should be named as a .tsv file, such as: SampleID_set1.tsv.

If using the Shiny app, you must specify the folder containing these SNP files. Multiple samples (with multiple files each) can be in the same folder. Additional files may be present in the folder and will be ignored by MixDeR.

MixDeR will divide the entire Kintelligence dataset into more manageable sets (organized by total SNP read depth) to run through EFM (ideal for best performance). The user may specify how many sets the program will use (see below); the default is 10 sets. If providing TSV files, the SNP should be divided into multiple sets and named accordingly (i.e. SampleID_set1.tsv). The user must then specify how many sets are provided so MixDeR knows how many files to process per sample.

The default is for MixDeR to use previously-created SNP sets, if present in the specified input folder. If this option is unselected, MixDeR will create new SNP sets files, overwriting any previously made files.

2. The sample manifest
   This file lists the Sample IDs of the files to run. The SampleID is extracted from the Sample Name field in the Sample Report. The columns names do not matter, just the order and both columns must be present even if no replicates are included. If a single sample is run, only the single ID needs to be in the first column, the second column should be left blank. If a second sample is to be run in replicate, the replicate ID should be listed in the second column.

SampleID	ReplicateID
Sample01a
Sample01a	Sample01b

Other files which may be required

Allele frequency file

MixDeR provides general population allele frequencies for Kintelligence SNPs from either 1000 Genomes Phase 3 dataset or the gnomAD v4 dataset. However, it is ideal to use allele frequencies derived from the population that closely matches the contributor(s) of interest. Therefore, MixDeR provides the user the opportunity to upload a different allele frequency file. EuroForMix requires the below format for allele frequency files, for all SNPs with each SNP as its own column:

Allele	rs6690515	rs424079	rs2055204
A	0.122837	0.64677	0.501441
C	NA	0.35323	NA
G	0.877163	NA	0.498559
T	NA	NA	NA

Given the difficulty of formatting the data as such, MixDeR will create this format for the user if the user provides the frequency data in a CSV file with the following format (NOTE: the column names MUST match below; the order of columns and additional columns will not affect it).

SNP	Ref	Alt	Ref_AF	Alt_AF
rs6690515	A	C	0.35323	0.64677
rs424079	T	C	0.122837	0.877163
rs2055204	G	A	0.501441	0.498559

MixDeR provides the option to use the same allele frequency file for both the major and minor contributor or select different allele frequency files for the major and minor contributor. If two different frequency files are selected, MixDeR will run EFM twice, once using the allele frequency file for the major contributor (and extracting the inferred genotypes for the major contributor from those results) and once using the allele frequency file for the minor contributor (and extracting the inferred genotypes for the minor contributor from those results).

Reference Genotypes

If calculating validation metrics or performing a conditioned deconvolution, the reference genotypes are required.

There are two options for providing reference genotypes. MixDeR accepts the UAS Sample Report, stored in a separate folder. A second option is to provide a single CSV file containing all references named EFM_references.csv with the following format:

Sample.Name	Marker	Allele1	Allele2
Sample01	rs12615742	T	C
Sample01	rs16885694	G	G

The user must provide the folder containing either the Sample Reports or the CSV reference file.
NOTE: MixDeR first searches for the CSV file in the provided folder. If there are additional wanted references not contained in this file, please remove the CSV file and run MixDeR again. MixDeR creates the CSV file containing genotypes from the Sample Reports within the provided folder.
________

Details

MixDeR has three modules:

1. EFM mixture deconvolution
2. Calculate validation metrics
3. Create GEDmatch PRO reports

EFM mixture deconvolution must be run at least once. If it’s been run previously, the other modules can be run using the existing deconvolution data.

MixDeR can either calculate validation metrics OR create the GEDmatch PRO reports during a single run, not both.

NOTE about the allele probability thresholds:
This workflow utilizes individual probabilities for each allele call from EFM. The reported allele 1 is the allele with the higher probability; allele 2 is the allele with the lower probability. When applying the allele 1 probability threshold, any SNP with a probability below the threshold will be removed completely from the dataset. When applying the allele 2 probability threshold to the remaining SNPs, if the probability is below the threshold, allele 2 is reported as the same allele as allele 1. If it is above, the allele will be reported as called. For example, if the genotype for SNP rs12615742 is C,T but the allele 2 probability is below the threshold, the SNP genotype will be reported as C,C. If it is above the threshold, the SNP genotype will be reported as C,T.

Running mixture deconvolution using EuroForMix

There are several options/settings to run EFM mixture deconvolution:
The type of mixture deconvolution analysis to perform (one or both can be selected at once):
* Unconditioned analysis
* Conditioned analysis
Allele Frequency Data: The user must choose which allele frequency data to use: 1000Genomes Phase 3 data, gnomAD v4 data, or upload a custom file. See above for more details about the format for uploading a custom AF file.
References to Condition on: IF running a conditioned analysis, once the reference folder has been uploaded, this dropdown menu will auto-populate containing the reference sample IDs. The user must select which references to condition on. As of now, MixDeR can only condition on a single reference sample. However, multiple references can be selected for conditioning; the conditioned analyses will be run separately.
Number of SNP Bins: The number of SNP sets for each sample. The default is 10.
Static Analytical Threshold: The minimum number of reads required for a SNP to be included (default = 10).
Dynamic Analytical Threshold: The percent of total SNP reads required for a SNP to. be included (default = 0.015 or 1.5%). (A quick note on the ATs: both the static and dynamic ATs are applied and the one producing the higher AT will be used in the EFM software).
Output Folder Name: The name of the folder created within the folder containing the original SNP files to store the generated output for the entire workflow.
Minimum Number of SNPs: The minimum number of SNPs

Calculating validation metrics

There are several options/settings to calculate the validation metrics:
Minimum Allele 1 Probability Threshold and Maximum Allele 1 Probability Threshold: The range of allele 1 probability thresholds for calculating the validation metrics, increasing in increments of 0.01 (i.e. if minimum is set to 0.5 and maximum is set to 1, will calculate metrics using a threshold of 0.5, 0.51, 0.52, 0.53, up to 1).
Minimum Allele 2 Probability Threshold and Maximum Allele 2 Probability Threshold: The range of allele 2 probability thresholds for calculating the validation metrics, increasing in increments of 0.01 (i.e. if minimum is set to 0.5 and maximum is set to 1, will calculate metrics using a threshold of 0.5, 0.51, 0.52, 0.53, up to 1).
Major Contributor Sample ID: The ID of the major contributor of the mixture. Once the reference folder is uploaded, this dropdown menu will auto-populate with the reference sample IDs.
Minor Contributor Sample ID: The ID of the minor contributor of the mixture. Once the reference folder is uploaded, this dropdown menu will auto-populate with the reference sample IDs.
Remove SNPs If Missing Either Allele?: If an allele 1 is inferred to be missing (reported as 99), that SNP will be automatically dropped from the final dataset. By default, if an allele 2 is inferred to be missing and the allele 2 probability is above the allele 2 probability threshold, the SNP is reported as homozygous for allele 1. However, selecting this option will result in dropping the SNP if the allele 2 probability of the missing allele 2 is above the allele 2 probability threshold, instead of reporting the SNP as homozygous for allele 1.

If calculating validation metrics, reference genotypes are required to calculate genotype accuracy. MixDerR will calculate the metrics for the range of allele 1 probability thresholds and allele 2 probability thresholds specified by the user. The final output file looks as such:

A1 cutoff	A2 cutoff	Total SNPs	N No Ref	N SNPs tested	N Genotypes Correct	Genotype Accuracy	Heterozygosity
0.99	0.01	9735	8	9727	9549	0.9817	0.456
0.99	0.02	9735	8	9727	9548	0.9815	0.456
0.99	0.03	9735	8	9727	9548	0.9815	0.456

Creating GEDmatch PRO Reports

Allele 1 Probability Threshold to create GEDmatch PRO Report and Allele 2 Probability Threshold to create GEDmatch PRO Report: The allele 1 and allele 2 probability thresholds. If the contributor is the major contributor to the mixture, MixDeR will apply the allele 1 and allele 2 probability thresholds UNLESS this results in a profile with fewer SNPs than the specified minimum (6000 is the default). If it does not meet this minimum, the top 6,000 SNPs (or whatever the user specifies as the minimum) with the highest allele 1 probabilities are used and then the allele 2 probability threshold is applied. For minor contributors, the default setting is that the minimum number of SNPs is automatically used and then the allele 2 probability threshold is applied. The option to apply the allele 1 probability threshold (similar in manner to the major contributor) can be applied.
Remove SNPs If Missing Either Allele?: If an allele 1 is inferred to be missing (reported as 99), that SNP will be automatically dropped from the final dataset. By default, if an allele 2 is inferred to be missing and the allele 2 probability is above the allele 2 probability threshold, the SNP is reported as homozygous for allele 1. However, selecting this option will result in dropping the SNP if the allele 2 probability of the missing allele 2 is above the allele 2 probability threshold, instead of reporting the SNP as homozygous for allele 1.

As a way to assist the analyst in evaluating the inferred genotypes of a mixture of unknown contributors, several metrics are calculated in this step for three different scenarios: (1) only the allele 2 probability threshold applied; (2) the allele 1 and allele 2 probability thresholds applied; and (3) the minimum number of SNPs used and the allele 2 probability threshold applied. For each dataset, the follow metrics are calculated: number of SNPs, mean allele 1 probabilities, median allele 1 probabilities, standard deviation (SD) of the allele 1 probabilities and heterozygosity. Below is an example of the table created by MixDeR in this step. A density plot of allele 1 probability thresholds is also created. In general, the more SNPs with higher allele 1 probabilities, the higher the accuracy of the inferred genotypes. However, determining exactly what qualifies as acceptable should be determined by individual labs.

Allele1 Threshold Applied	Allele2 Threshold Applied	Total SNPs	Mean A1 Prob	Median A1 Prob	SD A1 Prob	Heterozygosity
No	Yes	10024	0.9984	1	0.0096	0.4626
Yes	Yes	9718	0.9998	1	9.00E-04	0.4569
Minimum # of SNPs	Yes	6000	1	1	0	0.4495