lusSTR is a tool written in Python to convert NGS sequence data of forensic STR loci to different sequence representations (sequence bracketed form) and allele designations (CE allele, LUS/LUS+ alleles) for ease in downstream analyses. See the below section Converting STR sequences to other sequence representations and allele designations for more information.
Further, lusSTR can perform filtering and stutter identification using the CE allele, the LUS+ allele, or the bracketed sequence form for autosomal loci and create files for direct input into three probabilistic genotyping software packages, EuroForMix (CE and LUS+), MPSproto (NGS), and STRmix (CE and NGS).
lusSTR also processes SNP data from the Verogen ForenSeq and Kintelligence panels and create evidence and/or reference files for use in EFM. See the below section SNP Data Processing for more information.
This Python package has been written for use with either:
- ForenSeq Signature Prep panel
- 27 autosomal STR loci
- 24 Y-chromosome STR loci
- 7 X-chromosome STR loci
- 94 identity SNPs
- 22 phenotype (hair/eye color) SNPs
- 54 ancestry SNPs
- 2 phenotype and ancestry SNPs
- ForenSeq Kintelligence panel
- 10,230 SNPs for forensic genetic genealogy purposes
- Promega PowerSeq panel
- 22 autosomal STR loci
- 22 Y-chromosome loci
The package accomodates either the Sample Details Report/Phenotype Report/Sample Report from the ForenSeq Universal Analysis Software (UAS), STRait Razor v3 output, or GeneMarker v2.6 (*_strresults_filtered.csv output file; PowerSeq sequences only). If STRait Razor or GeneMarker output is provided, sequences are filtered to the UAS sequence region for conversion.
It is important to note: the most recent lusSTR version (version 0.9) utilizes the most recent STRait Razor configuration files as of December 2023 (ForenSeqv1.27.config and PowerSeqv2.31.config). If using STRait Razor data as input, please ensure you are using these config file versions when running STRait Razor v3s.
For best results, install from bioconda.
conda install -c bioconda lusstr
Contributors/developers who want to create a dedicated environment on their machine can do so:
conda create --name lusSTR -y python=3.10 pandas
git clone https://www.github.com/bioforensics/lusSTR.git
cd lusSTR
make devenv
lusSTR accomodates four different input formats:
(1) UAS Sample Details Report, UAS Sample Report, and UAS Phenotype Report (for SNP processing) in .xlsx format (a single file or directory containing multiple files)
(2) STRait Razor v3 output with one sample per file (a single file or directory containing multiple files)
(3) GeneMarker v2.6 output (*_strresults_filtered.csv; a single file or directory containing multiple files)
(4) Sample(s) sequences in CSV format; first four columns must be Locus, NumReads, Sequence, SampleID; Optional last two columns can be Project and Analysis IDs.
These individual sample files or directory of files must be specified in the config file (see below).
lusSTR utilizes the lusstr command to invoke various Snakemake workflows. The lusstr strs command invokes the STR analysis workflow while the lusstr snps command invokes the SNP analysis workflow. Please see below for further information on processing SNP data.
Running lusstr config creates a config file containing the default settings for the lusSTR STR analysis pipeline. The settings can be changed with command line arguments (see below) or by manually editing the config file. The default settings, along with their descriptions, are as follows:
analysis_software: uas (uas/straitrazor/genemarker); indicates the analysis software used prior to lusSTR
sex: False (True/False); include sex-chromosome STRs (invoke --sex flag)
samp_input: /path/to/input/directory/or/samples input directory or sample; if not provided, will be current working directory (indicate using --input path/to/dir )
output: lusstr_output output file/directory name (indicate using --out dir/sampleid e.g. --out test_030923)
kit: forenseq (forenseq/powerseq) (invoke the --powerseq flag if using PowerSeq data)
nocombine: False (True/False); do not combine identical sequences during the convert step, if using STRait Razor data. (invoke the --nocombine flag)
output_type: strmix (strmix/efm/mpsproto) (indicate using the --software flag)
profile_type: evidence (evidence/reference) (invoke --reference flag if creating a reference output file)
data_type: ngs (ce/ngs/lusplus) (indicate using the --str-type)
info: True (True/False); create allele information file (invoke --noinfo flag to not create the allele information file)
separate: False (True/False); for EFM/MPSproto only, if True will create individual files for samples; if False, will create one file with all samples (invoke --separate flag to separate EFM/MPSproto output files)
nofilters: False (True/False); skip all filtering steps but still creates EFM/MPSproto/STRmix output files (invoke --nofilters flag)
strand: uas (uas/forward); indicates the strand orientation in which to report the sequence in the final output table for STRmix NGS only (indicate using --strand)
One additional argument can be provided with lusstr config:
-w/-workdir sets the working directory (e.g. -w lusstr_files/) and all created files are stored in that directory.
Once the config file is created with all the desired settings, the STR workflow can be run. The config file must be located in the working directory.
The lusSTR STR workflow consists of three steps:
(1) format: formatting input
(2) convert: converting sequences to other sequence representations and allele designations
(3) filter: performing several filtering steps and creating appropriately formatted files for use in EuroForMix or STRmix.
Any or all steps can be run. In order to run all three steps, the following command can be used:
lusstr strs all
One additional argument can be provided, a working directory.
This working directory must contain the config file.
If not specified, the working directory is the current directory.
lusstr strs all -w lusstr_files/
Individual steps can also be run
lusstr strs format -w lusstr_files/
lusstr strs convert -w lusstr_files/
In order to run the convert step, the appropriately formatted .csv file containing the sequences normally created in the format step must be present in the working directory. See the below Formatting input for STR loci sequences section for specific information about that file (required columns, etc.).
If inputting data from either the UAS Sample Details Report, STRait Razor or GeneMarker output, the user must first invoke the format step to extract necessary information and format for the convert step.
The format command removes unnecessary rows/columns and outputs a table in CSV format containing the following columns:
- Locus
- Number of Reads observed with the specified sequence
- Sequence
- Sample ID
- Project ID (if provided)
- Analysis ID (if provided)
If including the sex chromosome loci as specified in the config file, the format command will output a second table for the sex loci with the same columns (*_sexloci.csv).
The convert step produces a tab-delineated table with the following columns:
- Sample ID
- Project ID (if provided)
- Analysis ID (if provided)
- Locus
- UAS Output sequence: can be forward or reverse strand
- Forward strand sequence: will be same as UAS Output sequence for those loci reported on forward strand
- UAS Output Bracketed notation: Bracketed sequence form for the reported UAS sequence output (will be same for those loci which report the forward strand)
- Forward Strand Bracketed notation: Bracketed notation for forward strand sequence
- CE allele: common length-based CE allele designation (also called the repeat unit, or RU, allele)
- LUS: Longest uninterrupted stretch
- LUS+: Notation combining multiple allele designations including CE, LUS, secondary motif (if applicable) and tertiary motif (if applicable)
- Reads: number of reads observed with the specified sequence
If including the sex chromosome loci as specified in the config file, a second table with the above columns for the sex chromosome loci will be outputted as well.
If STRait Razor or GeneMarker data is specified, several additional processes occur with the convert step:
- The full sequences are filtered to the UAS region before the translation step. The number of bases to remove is determined based on the specified kit.
- Once the sequences are filtered to the UAS region, any duplicated sequences are removed and their reads are summed in with the remaining sequence
Readscolumn. NOTE: This step can be skipped with thenocombinesetting in the config file.
Further, a second table (labeled as *_flanks.txt) containing information related to the flanking sequences surrounding the UAS sequence region is also produced with the following columns:
- Sample ID
- Project ID
- Analysis ID (same as Project ID)
- Locus
- Reads: number of reads observed for the specified sequence
- Length-based (CE) Allele
- Full Sequence
- 5' Flanking Sequence Bracketed Notation
- UAS Region Sequence Bracketed Notation (same as column
UAS Output Bracketed Notationin the main table) - 3' Flanking Sequence Bracketed Notation
- Potential Issues (such as: Possible indel or partial sequence)
The Potential Issues column in this report is to draw attention to potential problem sequences (due to perhaps an indel or partial sequence) and requires the attention of the user to further evaluate the sequence for its authenticity.
Individual marker plots displaying reads vs. CE allele are created in PDF format. They are stored in the MarkerPlots/ directory of the working directory:
- The first page of the PDF file contains the marker plots using a custom y-axis scale (i.e. the y-axis is scaled for each individual marker).
- The second page of the PDF file contains the marker plots using the same (i.e. the maximum) y-axis scale.
- A separate PDF file for the X- & Y-STRs are created, if specified.
The filter step provides the opportunity to filter sequences using thresholds such as:
- Detection threshold (both static and dynamic)
- Analytical threshold (both static and dynamic)
- Same size threshold (dynamic)
Custom static and dynamic thresholds for each locus are stored in the filters.json file. This file should be updated to utilize validated thresholds for individual labs.
In addition, stutter alleles can be identified using the info setting in the config file. This creates a separate file containing information about each allele, including an allele classification (real allele, stutter or BelowAT). Stutter alleles are classified as either -1 stutter, -2 stutter, or +1 stutter. For these stutter alleles, the stuttering allele is reported along with the percent stutter (# of reads for that allele/# of reads for stuttering allele). In instances where a stutter allele could be multiple different types of stutter, all potential designations will be reported as such: -1 stutter/-2 stutter, -1 stutter/+1 stutter, or -2 stutter/+1 stutter. No percent stutter is calculated for these alleles. If a sequence is identified as noise, the percent noise is calculated (# of reads for that sequence/total locus reads).
Each locus is checked for containing greater than 2 alleles (indicating a potential mixture) and for intralocus imbalance. If either are identified, a separate file (Flagged_Loci.csv) is created, containing the SampleID, Locus and either >2Alleles or IntraLocusImbalance.
When using STRmix data, the data type can be specified using the data-type setting as either ce, ngs or lusplus (default is ngs). If ngs or lusplus is specified, the same size filter is applied following the stutter filter. Further, the columns and column names in the output file differ based on the data type.
Finally, output files are created for direct use in EuroForMix (EFM), MPSproto or STRmix. If EFM or MPSproto is specified, a single file is created containing all samples in the input file (however, separate output files for each sample can be created with the separate setting specified in the config file). If STRmix is specified, a directory containing files for each individual sample is created. The profile-type setting allows for the creation of either a reference or evidence profile. Both EuroForMix/MPSproto and STRmix require different formatting depending on the type of sample.
lusSTR is able to process SNPs derived from the ForenSeq Signature Prep assay and the ForenSeq Kintelligence assay. SNPs from the ForenSeq Signature Prep assay could be analyzed using either the Verogen UAS or STRait Razor. SNPs from the ForenSeq Kintelligence assay must first be analyzed using the UAS.
Running lusstr config --snps creates a config file containing the default settings for the lusSTR SNP workflow. The settings can be changed with command line arguments (see below) or by manually editing the config file. The default settings, along with their descriptions, are as follows:
analysis_software: uas (uas/straitrazor); indicates the analysis software used prior to lusSTR
samp_input: /path/to/input/directory/or/samples input directory or sample; if not provided, will be current working directory (indicate using --input path/to/dir)
output: lusstr_output output file/directory name; (indicate using --out dir/sampleid e.g. --out test_030923)
kit: sigprep (sigprep/kintelligence) (invoke using the --kintelligence flag if using Kintelligence data)
types: all choices are "all", "i" (identity SNPs only), "p" (phenotype only), "a" (ancestry only) or any combination (indicate using the --snp-type e.g. --snp-type i, p)
nofilter: False (True/False); if no filtering is desired at the format step; if False, will remove any allele designated as Not Typed (invoke using the --nofiltering)
strand: forward (forward/uas); indicates which orientation to report the alleles for the SigPrep SNPs; uas indicates the orientation as reported by the UAS or the forward strand
references: None; list IDs of the samples to be run as references in EFM; default is no reference samples
separate: False (True/False); if want to separate samples into individual files for use in EFM
thresh: 0.03; Analytical threshold value
One additional argument can be provided with lusstr config --snps:
-w/-workdir sets the working directory (e.g. -w lusstr_files/) and all created files are stored in that directory.
Once the config file is created with all the desired settings, the SNP workflow can be run. The config file must be located in the working directory.
The lusSTR SNP workflow consists of two steps:
(1) format: formatting input and calling alleles if using STRait Razor data
(2) convert: applying analytical threshold and converting data to correct format for input into EuroForMix
Either the first step, format, or both steps can be run. In order to run both steps, the following command can be used:
lusstr snps all
One additional argument can be provided, a working directory.
This working directory must contain the config file.
The default working directory is the current directory.
lusstr snps all -w lusstr_files/
Individual format step can also be run
lusstr snps format -w lusstr_files/
In order to run the all step, the appropriately formatted .csv file containing the sequences normally created in the format step must be present in the working directory. See the below Usage section for specific information about that file (required columns, etc.).
If inputting data from either the UAS Sample Details Report/Phenotype Report/Sample Report or STRait Razor output, the user must first invoke the format step to extract necessary information and format for the final convert step.
The format command removes unnecessary rows/columns and outputs a table in CSV format containing the following columns:
- Sample ID
- Project ID
- Analysis ID
- SNP (rsID)
- Reads
- Forward Strand Allele
- UAS orientation Allele
- Type (ancestry/identity/phenotype/kintelligence)
- Issues
This final step will convert the table generated in the format step into the correct format for use in EuroForMix. An analytical threshold can be applied (this is especially useful for data analyzed using STRait Razor) in this step.
If any samples are to be used as references, their IDs can be provided in the config file to create a separate file appropriately formatted for use as reference profiles in EFM. Any samples not specified as references are assumed to be evidence samples and will be formatted as such.
There is the option to create separate evidence files for each sample (as specified in the config file); this is especially useful for Kintelligence profiles given their larger size.
This command also changes the alleles to numeric (A=1, C=2, G=3, T=4)
lusSTR is still under development and any suggestions/issues found are welcome!