Forensic Science International: Genetics 33 (2018) 117–128

Contents lists available at ScienceDirect

Forensic Science International: Genetics

journal homepage: www.elsevier.com/locate/fsigen

Review article

A review of bioinformatic methods for forensic DNA analyses T

a,⁎ b
Yao-Yuan Liu , SallyAnn Harbison
a
Forensic Science Program, School of Chemical Sciences, University of Auckland, 38 Princes Street, Auckland, 1010, New Zealand
b
Institute of Environmental Science and Research Limited, Private Bag 92021, Auckland, 1142, New Zealand

A R T I C L E I N F O A B S T R A C T

Keywords: Short tandem repeats, single nucleotide polymorphisms, and whole mitochondrial analyses are three classes of
Bioinformatics markers which will play an important role in the future of forensic DNA typing. The arrival of massively parallel
Forensic science sequencing platforms in forensic science reveals new information such as insights into the complexity and
Massively parallel sequencing variability of the markers that were previously unseen, along with amounts of data too immense for analyses by
manual means. Along with the sequencing chemistries employed, bioinformatic methods are required to process
and interpret this new and extensive data. As more is learnt about the use of these new technologies for forensic
applications, development and standardization of efficient, favourable tools for each stage of data processing is
being carried out, and faster, more accurate methods that improve on the original approaches have been de-
veloped. As forensic laboratories search for the optimal pipeline of tools, sequencer manufacturers have in-
corporated pipelines into sequencer software to make analyses convenient. This review explores the current state
of bioinformatic methods and tools used for the analyses of forensic markers sequenced on the massively parallel
sequencing (MPS) platforms currently most widely used.

1. Introduction
In recent years, the forensic community has been exploring the use
of massively parallel sequencing (MPS) for DNA analysis [1–7]. As the
cost of MPS platforms became more affordable, useful information ex-
isting in parts of DNA previously overlooked or deemed uneconomical
to examine could be investigated. Børsting and Morling have provided a
detailed introduction to MPS forensic analysis [8]. An area of great
focus in forensic MPS studies is in data processing, where methods and
techniques are being explored for the analyses of the large amounts of
data produced during an MPS run. Forensic science has inherited
bioinformatic workflows and methods used in other fields of DNA sci-
ence [9–12]. These workflows were further developed and/or stimu-
lated the production of new tools for forensic science [13–17].
Current DNA markers of forensic interest are short tandem repeats
(STRs), single nucleotide polymorphisms (SNPs) and polymorphisms in
the whole mitochondrial genome (mtDNA)[18,19]. These markers
provide different types of information, and as the characteristics of each
marker type are different, specific pipelines and tools have been de-
veloped for each marker type.
Other types of forensic DNA markers have also been described, such
as indels (for example [20–22]), epigenetic methylation markers (for
example [23–27]) and Alu repetitive elements (for example [28–31]).
In addition, metagenomic approaches to forensic evidence [32–34] as
well as other non-human genetic evidence (for a review see [35]) are
gaining attention as MPS technology facilitates their adoption. For the
convenience of the analyst, manufacturers of MPS platforms have
packaged analysis tools with their machines in the form of plugins
within analytical software frameworks. These include the MiSeq Re-
porter/ForenSeq Universal Analysis Software bundled with the MiSeq
(Illumina; San Diego, Ca., USA, https://www.illumina.com), and the
Torrent Suite bundled with the Ion Torrent PGM (Thermo Fisher Sci-
entific, South San Francisco, CA, USA, https://www.thermofisher.com).
Bioinformatic pipelines provided by the manufacturers of MPS
platforms offer convenience at the expense of variety and flexibility of a
hand-assembled pipeline. Manufacturer-provided solutions are indeed
useful and have been widely used by the forensic science community. In
this review, they are described along with open-source pipelines in their
relevant marker sections. In this rapidly changing environment, se-
quence analyses software remains in continued development and ver-
sions of the software described in published papers may be outdated by
the time of reading.
This review describes the main marker systems currently used, or
considered to be used, in forensic bioinformatics and introduces
bioinformatic solutions for data processing and analyses. Bioinformatics
tools most relevant to forensic practice are presented, and review ar-
ticles are provided for further information where these are considered
helpful, however, it is not possible to include all the possible tools

⁎
Corresponding author.
E-mail address: alex.liu@esr.cri.nz (Y.-Y. Liu).

https://doi.org/10.1016/j.fsigen.2017.12.005
Received 13 September 2017; Received in revised form 30 November 2017; Accepted 10 December 2017
Available online 12 December 2017
1872-4973/ © 2017 Elsevier B.V. All rights reserved.
Y.-Y. Liu, S. Harbison
existing in the sphere of biological computation. Forensic DNA data-
bases will also be introduced.
In addition to population sequence databases such as the genome/
exome Aggregation Databases (genomAD, http://gnomad.
broadinstitute.org, and ExAC, http://exac.broadinstitute.org/), and
the 1000 Genomes Project [36,37], the forensic community now has
access to marker-specific databases such as EMPOP [38] for mtDNA,
ALFRED [39] and STRidER [40] for SNP and STR allele frequencies
respectively, and STRseq [41] for STR sequence variants.
This review does not cover the subsequent interpretations of STR
profiles, such as the resolution of the components of a mixture or the
determination of a match.
2. Data preparation
Before the amplified DNA fragments are sequenced, short sequences
are appended to the DNA during library preparation to facilitate the
sequencing process [8]. As such, the raw, digital form of a DNA read
contains the sequence of interest as well as the additional appended
sequences. These include the PCR primers that flank the sequence of
interest, a barcode (index) sequence for recognition of a sample’s
origin, and adapter sequences at the ends required to attach and im-
mobilize each library to a solid surface for sequencing. These sequence
additions are later removed to reduce the sequence of interest to its
original state. MPS platforms such as the Ion Torrent PGM, and the
Illumina MiSeq produce adapter-trimmed, demultiplexed FASTQ [42]
files, This means each sequence read is sorted and assigned by barcode
to their sample of origin. Each such FASTQ file contains the DNA se-
quences and their quality scores.
Data preparation, or data preprocessing, are the first steps in an
MPS data analysis pipeline. This includes quality assessment/control
steps, and for some applications, an alignment step (Fig. 1).
For a review on general tools for variant analysis on MPS data,
Pabinger et al. [43] provide a survey of several quality assessment,
alignment, and variant identification tools not covered in this review.
In most formats, the DNA data can be visualized and explored with
several software options. Examples of these are the Integrative
Genomics Viewer (IGV, http://software.broadinstitute.org/software/
igv/home), Geneious (https://www.geneious.com/), and NextGENe
(http://www.softgenetics.com/NextGENe.php).
2.1. Data quality assessment and control
Since MPS instruments cannot identify individual bases with
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Forensic Science International: Genetics 33 (2018) 117–128
absolute certainty, quality scores are assigned to each base by the se-
quencer as a metric of the probability of an incorrect basecall. In other
words, the software calculates the probability of an incorrect base call.
The lower the probability, the more confident the call. This is because
inclusion of poor quality data may lead to noise or systemic errors
during analysis and affect the results.
Quality control includes the assessment of the data quality and
subsequent removal of poor quality information.
Visualization of the data quality may be useful to check the success
of the run and overall base quality trends to identify any potential
problems with the data. FastQC (http://www.bioinformatics.bbsrc.ac.
uk/projects/fastqc) developed by the Babraham Bioinformatics group,
provides statistics and graphical displays of the per base sequence
quality, read lengths and other helpful metrics from FASTQ and BAM/
SAM files (described below). SolexaQA is another tool for quality
analysis and visualization, but also packaged with quality control tools
such as a read trimmer and a length-based filter [44]. This tool accepts
FASTQ files.
Quality control tools such as quality trimming and quality filtering
remove poor quality bases using Phred quality scores [42]. Read
trimming, the removal of low-quality sequence tracts from a read, is
useful for removing poor quality bases from the ends of reads, while
read filtering is useful for removing entire reads of overall poor quality.
The FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit) contains
both a quality filter and a quality trimmer along with many other tools.
As base quality drops towards the end of a sequence read, lower
quality bases at the 3′ end of reads are removed by default in the
Torrent Suite Software using a sliding window approach, where the
window size is 30 bases and the trimming threshold is a q score of 15.
Reads that are short (less than eight base pairs) or adapter dimers are
also removed as part of read filtering (https://ts-pgm.epigenetic.ru/ion-
docs/Technical-Note—Filtering-and-Trimming_6455370.html).
Illumina data, on the contrary, are not quality trimmed or filtered by
default, and these steps would need to be manually included in the
analysis pipeline if necessary.
2.2. The general-purpose pipeline: align and variant call
For the majority of sequence studies, alignment to a reference
genome and variant calling form the core of a general workflow for
genetic variant examination.
Pipelines that adopt the alignment approach [2,7,45] include an
alignment step in which reads are mapped/aligned to a reference
genome creating an alignment file. Note that the International Society
Fig. 1. Sequences added to DNA for sequencing, and
their bioinformatic removal.
Y.-Y. Liu, S. Harbison
for Forensic Genetics (ISFG) recommends using the most recent build of
the human reference genome hg38, which includes the mitochondrial
reference standard, rCRS. Variant calling is then performed on the
alignment file to determine the genotype of each base position of in-
terest. This approach has been used in examining SNPs, mtDNA, and
STRs, but other strategies exist, such as the sequence-search approach
for STRs and SNPs.
Alignment pipelines mainly consist of a short read aligner and a
variant caller. Although seemingly simple in construction, different
combinations of these components can yield varying results [12,46,47].
For more information on alignment-variant calling pipelines,
Cornish et al. [46] and Hwang et al. [47] have provided detailed
comparisons of pipelines constructed from different tools.
2.2.1. Alignment
Read aligners map reads to a reference genome. There are many
aligners available, such as Bowtie 2 [48], Novoalign (http://novocraft.
com), and BWA-MEM [49]. Bowtie and Burrows-Wheeler Aligner
(BWA) are very efficient [50] and have been widely used.
Some aligners are better suited to data produced on particular
platforms due to the platforms’ characteristic data patterns. For ex-
ample, BWA is suitable for Illumina data, which contain mainly sub-
stitution errors, and TMAP (https://github.com/iontorrent/TS/tree/
master/Analysis/TMAP) is more suitable for Ion Torrent reads which
contain predominantly indel errors [51].
For more information on aligners, Fonsenca et al. [52] performed a
survey of over 60 aligners and present an overview of their character-
istics.
The output of read aligners is an alignment file in SAM (Sequence
Alignment/Map) format [53]. The SAM/BAM formats are the industry
standard for alignments. SAM files contain human-readable text, with
each row within the file detailing an alignment of a read to the re-
ference genome. For faster processing by computers, SAM is converted
to BAM (Binary Alignment/Map), a binary form of the same informa-
tion. This is done using SAMtools, the software package for parsing and
manipulating SAM/BAM alignments [53].
BAM files can be sorted and indexed to further improve reading
efficiency by downstream tools. BAM file sorting orders the reads based
on their position in the reference, done using the Picard Tools (https://
broadinstitute.github.io/picard/) SortSam module, or the sort function
in SAMtools. Indexing the BAM file with the SAMtools’ index function
creates an index file (.bai), which allows for the more efficient reading
of the BAM file.
Local realignment can be done to realign misaligned reads, which
may produce bioinformatic artefacts that resemble SNPs and indels. The
Genome Analysis Toolkit (GATK) contains the modules
RealignerTargetCreator and IndelRealigner for this purpose [11,54].
Tools for STR local realignment are described in Section 4.3.
2.2.2. Variant calling
Variant callers identify the differences between the sample and a
reference sequence and typically take alignment SAM/BAM files as
input and generate a Variant Call Format (VCF) file [55] (its binary
counterpart is the BCF file) which is used for variant calling and
downstream analyses and interpretation. BCFtools is a software toolset
for variant calling which reads and writes VCF and BCF files. The GATK,
and Freebayes are also common variant callers.
Variant calling with BCFtools (included as part of the SAMtools
package) involves calling the mpileup command to produce a BCF file
and computes the genotype likelihood at each genomic position. Then
the bcftools command is called to calculate the genotype and call the
variants, producing a VCF file. Variant filtering, subject to analysis, is
applied by running the vcfutils.pl script to remove false positives or
low-quality calls.
The GATK contains a Haplotype caller (which replaced the Unified
Genotyper) which performs a local re-assembly, calculates haplotype
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likelihood, and then determines the most likely genotype of SNPs and
indels.
3. SNPs
3.1. Current state
SNPs are versatile markers, capable of providing additional in-
formation from forensic samples such as ancestry and externally visible
characteristics, alongside STR profiling which is the current standard
for human identification.
In forensic science, the single nucleotide changes that usefully in-
form ancestry or phenotype are typically SNPs, common to at least 1%
of the population being studied, whilst those found in flanking regions
are more often described as novel and are therefore considered rare and
should be called SNVs. Over time as more forensic MPS studies are
conducted, some SNVs may be found to be more properly termed SNPs,
at least in some populations. For the purposes of this review (and most
often in the literature) all of these single nucleotide differences are
referred to as SNPs.
Genome-wide association studies and the HapMap project [56]
generated an immense amount of SNP data. Based on the type of in-
formation that can be inferred, forensically-relevant SNPs were selected
and classed into four categories: Identity-informative (IISNPs) for in-
dividualization, lineage-informative SNPs (LISNPs) for kinship haplo-
typing, ancestry-informative SNPs (AISNPs) for biogeographical an-
cestry inference, and phenotypic-informative SNPs (PISNPs) for
external phenotype prediction [57].
A wide array of techniques and technologies exist for SNP geno-
typing and these are well described in Sobrino’s review [58].
To date, forensically relevant SNPs have been mainly genotyped
using the SNaPshot technique, which employs electrophoresis and
fluorescence detection of SNPs [58]. In brief, a primer targeting the SNP
anneals adjacent to the SNP position then is extended by a DNA poly-
merase to incorporate a fluorescently labelled dNTP at the SNP posi-
tion. The fluorescent colour is then detected by capillary electrophor-
esis (CE) to identify the SNP [59].
A wide range of SNaPshot assays are used for routine SNP testing
and these are described in Mehta’s review [60]. Other SNP genotyping
methods such as the Genplex system from Applied Biosystems have
been used (for example [61]) however these methods are limited by the
number of SNPs that can be tested. MPS offers a much larger number of
SNPs that can be genotyped concurrently compared with earlier assays,
as well as the ability to run multiple samples together, detection of
every nucleotide in the amplicon, and the pooling of multiple SNaPshot
panels [62].
There is an ongoing effort in forensic SNP panel development, done
usually by scrutinising freely available online data such as the 1000
genomes project, assessing SNP performance from different panels,
combining them into a panel and testing against relevant populations;
the variety of such panels span populations, information types, and
autosomal and sex chromosomes [63–70].
The replacement of STRs by SNPs as the dominant forensic marker
has also been debated [71]. These single-base markers make the ex-
traction and amplification from degraded DNA samples easier and are
less prone to stochastic effects. SNPs also have lower mutation rates
than STRs and may associate with phenotypic traits, thus making them
useful for a variety of applications, and being non-tandem-repeat
markers, the lack of stutter artefacts vastly improves mixture inter-
pretation, including an assessment of the number of potential con-
tributors. The main disadvantage of SNPs is the higher number of loci
required in order to match the discriminatory power of STR assays and
the difficulty in mixture analysis due to the limited polymorphism
(usually bi-allelic) of the SNP alleles [71]. These disadvantages may be
overcome using MPS techniques and analysing larger numbers of SNPs.
Microhaplotypes are loci with two or more SNPs that occur in a
Y.-Y. Liu, S. Harbison
short segment of DNA within the length of a MPS read, and collectively
define a multiallelic locus [72,73]. These microhaplotype loci can
provide forensic information for identification, lineage and biogeo-
graphic ancestry. These markers can be used for mixture deconvolution,
and because they are unaffected by PCR stutter may be more robust
than the STRs. Kidd et al. have identified a collection of microhaplotype
loci and estimated their allele frequencies on 83 different populations,
demonstrating their superiority over individual SNPs, while fulfilling
the forensic objectives of STRs for discrimination between individuals
[74].
SNP reporting is not complicated by nomenclature issues, but
strandedness can cause issues in interpretation, where one allele is re-
ported for each chromosome and passed to an analysis tool. Most online
prediction tools, such as Snipper [75], are sensitive to the input se-
quence of the bases, and the input of the SNP on the wrong strand
effectively functions as a base miscall and leads to a compromised
analysis. The analyst should be aware of this when using SNP analysis
tools. It is notable that the Ion Torrent reports all SNPs in the forward
strand, while the MiSeq FGx reports SNPs consistent with dbSNP. Illu-
mina has devised a method for strand designation of ambiguous and
non-ambiguous SNPs: (https://www.illumina.com/documents/
products/technotes/technote_topbot.pdf).
3.2. Workflows and pipelines
3.2.1. Data processing
Processing of SNP MPS data is traditionally done using alignment
pipelines such as those described in Section 2.2.1, mainly consisting of
an aligner and a variant caller. However, sequencer-specific software
solutions have become popular and have been used in many published
studies [70,76,77].
Pipelines used in Ion PGM forensic SNP studies with the Torrent
Suite v3.0 involve the alignment of reads to hg19 using the Torrent
Mapping Alignment Program (TMAP), which produces a BAM align-
ment, and subsequent variant calling is done using the Torrent Variant
Caller v3.0 with default parameters, which produces a VCF file [62].
The HID_SNP_Genotyper is another Torrent Suite plugin based on
the Torrent Variant Caller. It performs SNP calling and outputs a VCF
file and a text file containing the SNP genotypes. A HID Genotyper
Report is generated that summarizes the data and analysis in a gra-
phical user interface which can be viewed on the Torrent Server. This
plugin is used in many studies [7,63,70,76,78].
On the MiSeq FGx, the bundled analysis software is called the
ForenSeq Universal Analysis Software (UAS). UAS performs alignment,
allele calling, genotyping, and generates a web-based report that can be
viewed on the server in a graphical user interface. The UAS has been
used in some studies [77,79,80].
Although being primarily STR analysis tools, STRait Razor [15] and
MyFLq [14] can be configured to extract SNP regions from a FASTQ file
without the need for alignment. The sequence-search approach used in
these tools for STRs extraction can be applied to SNPs, which involves
scanning the sequence data file for flanking sequences adjacent to the
SNPs. These tools are described in Section 4.2.
3.2.2. Databases
After SNPs are identified, a number of databases and online analysis
tools can be used for interpretation of the genotypes. Many of these
interactive tools make predictions based on population datasets. For
example, Snipper (http://mathgene.usc.es/snipper/) [75] is an online
SNP-Indel classification tool where users can choose a marker set, the
population groups, and a classifier algorithm. The user manually strings
together a collection of ancestry-informative SNP genotypes in a given
order and feeds the string into Snipper. The classification gives a pre-
diction of the population group and a percentage of possible admixture.
For example, using the test example SNP string on the site, the results
suggest that the individual is most likely European 89.24%.
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The multiple-individuals toolset in Snipper performs ancestry clas-
sification and accepts input data from multiple individuals in a
spreadsheet format. The user is freed from the fixed SNP input order
and can add SNP information for a customized analysis. (http://
mathgene.usc.es/snipper/analysismultipleprofiles.html). HIrisPlex
[65] is a validated set of SNP markers used to predict hair and eye
colour. The HIrisPlex website (http://hirisplex.erasmusmc.nl/) con-
tains tools that can be used to make predictions; the hair and eye colour
prediction from SNP alleles are produced using a Multinomial Logistic
Regression model trained on population data [81,82].
For SNP information, STRbase [83] contains a page on forensic
SNPs with a collection of reference materials. It includes a collection of
SNPs and available SNP assays, information on the varieties of SNPs,
typing technologies, and general SNP information.
The Allele Frequency Database (ALFRED) is an online and freely
accessible database containing allele frequencies of anthropologically
defined populations [39]. Its emphasis on population-based allele fre-
quencies sets it apart from databases serving as general catalogues of
polymorphisms such as dbSNP [84]. ALFRED data is used by the online
tool Forensic Research/Reference on Genetics knowledge base (FROG-
kb), which aims to make allele frequency data more accessible and
useful in a forensic setting. FROG-kb contains tools to compare user
data to allele frequencies in populations, with a focus on identification,
ancestry, and phenotype SNP types [85].
Ideally, global population sets could be used for interpretation, but
in practice, it is most likely that laboratories will develop independent,
local databases reflective of local populations and in keeping with na-
tional ethical and data-sharing legislation. Of note reference datasets
for the major MPS ancestry SNP sets are available through the Snipper
website (http://mathgene.usc.es/snipper/). Accessible, appropriately
validated online or on-instrument resources and tools will become an
important consideration for laboratories.
4. STRs
4.1. Current state
Short tandem repeats (STRs) are the standard marker type currently
used in forensic DNA profiling. STR alleles are typed by length using CE
methodology.
The methodology of STR typing by CE is well developed [86], and
many commercial STR PCR amplification kits are available, such as the
GlobalFiler™ PCR amplification kit from ThermoFisher Scientific [87],
the PowerPlex® 21 system from Promega Corporation [88], and the
Investigator 24 plex kit from Qiagen [89].
As MPS is becoming more widely adopted, STR typing by MPS is
considered to be feasible and has distinct advantages over CE, such as
the ability to detect STR sequence variations and the larger number of
DNA markers that can be multiplexed together.
Recently, MPS has been used in numerous studies of STRs including
the choice and evaluation of multiplex systems [5,90–92], the devel-
opment of statistical methods for interpretation [93], proof of concept
for simultaneous analysis of STRs with mRNAs for tissue identification
[94], improved resolution of parental cases [79], and the investigation
of STR sequence variations [95–98].
Concurrent with MPS studies of STRs were innovations in STR data
analyses methods and software tools. These include lobSTR [99],
STRait Razor [15,100,101], TSSV [102], the MyFLq framework [14],
STRinNGS [16], SEQ Mapper [103], and FDStools [13].
In the study of STRs, the nomenclature of STRs have been the
subject of ongoing discussions. These discussions follow from reports of
the extensive sequence variation within STR repeats that has been
found, flanking region polymorphisms found and requirements for
compatibility between laboratories and within laboratories for DNA
databasing of DNA sequence profiles.
As MPS generates sequence information as well as length, there has
Y.-Y. Liu, S. Harbison
been a search for a format that best conveys the sequence information
with all its variations, while maintaining compatibility with current CE
databases.
One proposed format contains the combined elements of STR locus
name, CE allele length, chromosome coordinates of the repeat motif in
the reference genome, the sequence with repeats in concatenated
bracket form, and variations outside of the STR [104]. An example from
this paper is as follows:
D13S317-CE12−chr1 3-GRC h37-g.82 .722 .160:82 .72
2.223−TATC[13] AATC[1] ATCT[3]−x. 136G > A
However, complications exist beyond the formulation of a STR no-
menclature. An example of the challenges in the creation of a new
system is the historical designation of STRs for use in CE, which were
not fully consistent, examples being the strand for repeat region des-
ignation and the inclusion of incomplete repeat sequences towards the
counting of repeat units in an allele [104]. This requires further work in
re-defining the STR regions.
The International Society for Forensic Genetics (ISFG) has a com-
pilation of suggestions and insights into the matter [105], and more
recently, the STR Sequencing Project (STRSeq) was initiated to collect
and curate STR sequence data to facilitate the standardization of STR
sequence nomenclature [41].
Suggestions include the use of a comprehensive format for in-
formation and backward-compatibility with CE, as well as a simple
format for easier communication. STR records in databases should be in
the form of complete sequence strings instead of shortened nomen-
clatures, so issues that arise from notation may be mitigated. To in-
crease discrimination, flanking region variation in the genome should
also be included in the database.
4.2. Workflows and pipelines
Following pre-processing and quality assessment of the raw MPS
data, STRs are detected, identified, and extracted.
Many methods of STR detection and identification have been de-
vised, giving rise to many pipelines based on different approaches
(Table 1). A few studies have performed data analysis using more than
one pipeline to test concordance and reliability [95,106].
To streamline STR data analysis, MPS machine manufacturers have
assembled and incorporated STR analysis pipelines into their software
solutions. These sequencer software such as the MiSeq ForenSeq UAS
and the Torrent Suite HID_STR_Genotyper plugin have been used in a
Table 1
Freely available STR analysis tools.
Tool name Intended platforms
loBSTRv4 Linux
Mac
Curoverse (online)
RepeatSeq Linux
STRviper Most platforms, as it is written in Java
CoalescentSTR Statistical method available in paper
STRait Razor v1.0-v2s Linux
STRait Razor v3 Windows
Mac
Linux
MyFLq Linux
Illumina BaseSpace (online)
Tool webpage (Online)
STRinNGS Linux
SEQ Mapper Online tool
TSSV Linux
Forensic Science International: Genetics 33 (2018) 117–128
range of STR studies [96,107,108].
4.2.1. Alignment approach
One of the earliest STR detection and identification approaches was
to align the reads to a reference genome and extract the STRs by their
locations on the alignment.
Bornman et al. developed a method to align reads to a FASTA re-
ference containing just the STR loci [2]. This took advantage of the fact
that forensic STR analyses were traditionally accomplished using am-
plicon sequencing instead of whole genome sequencing.
These reference files contain a concatenated STR sequence for each
locus and their upstream and downstream flanking sequences. The
alignment was done using Bowtie, using ungapped alignment to keep
the repeat sequences of the reads intact. Picard (http://broadinstitute.
github.io/picard/) and BedTools were used to convert the BAM files to
a Browser Extensible Data (BED) format that contains the start and end
positions of each read, then a custom script in R was used to filter out
reads that do not span the entirety of the STR and its flanks. Alleles
were called based on the number of reads, using a heuristic decision
model. The components of the pipeline are interchangeable with those
of a similar nature, such as swapping the tool Bowtie for BWA for the
alignment process [106].
lobSTR [99] is a STR extraction tool, a self-contained pipeline that
detects the presence of STRs on reads by repeat stretches. Finding the
STR stretch, STR locus flanks are aligned to the left and right side of the
stretch to identify the locus and reveal the number of repeat units. A
statistical model is used to call the loci alleles.
This approach was much faster and precise than ungapped align-
ment with short-read aligners [99]. However, shortcomings that were
identified were its ability to only analyse 2-6-mer simple motif repeat
loci, which make complex repeats difficult to analyse [100] and its lack
of awareness of homopolymer runs [9]. lobSTR has since been updated
to version 4.0.6. The approach introduced by lobSTR spawned in-
vestigations into two intertwined details of its workings, the correct
alignment of the reads to the reference and accurate estimation of re-
peat numbers.
Due to sequence variations that may differ from the reference allele
in the lobSTR database, and sequencing errors in STR sequences, initial
alignment may not place reads in the optimal locations, leading to
miscalled variants. Realignment tools can be used to revise misaligned
reads at STR loci. The changes made to the tool can be found at: http://
lobstr.teamerlich.org/changelog.html.
Input formats Output formats Reference
FASTQ/FASTA BAM alignment file [99]
BAM VCF file
A text file describing the most likely alleles.
BAM VCF file or two custom output formats [9]
FASTA + region file
BAM/SAM STRV file containing allele calls, convertible to VCF [111]
VCF (with scripts)
[112]
FASTQ/FASTA Allsequences.txt (All found sequences with read coverage) [101]
rawSTRcalls.txt (read counts of known alleles)
FASTQ/FASTA Allsequences.txt [15]
FASTQ XML files with alleles found and figures [14,116]
FASTQ PDF file with STR profile [16]
BAM A results file to be analysed with R
VCF
FASTA Web summary report [103]
FASTQ/FASTA Overview report of general statistics and identified alleles [102]
+reference alleles in CSV format
121
Y.-Y. Liu, S. Harbison
ReviSTER [109] is an automated pipeline for local realignment. It
first maps all the reads to the reference then generates a temporary
reference from the consensus. The temporary reference is used as the
reference for re-alignment. The reads mapped to the temporary re-
ference are then moved back to the original reference sequence.
STR-realigner [110] is another realignment tool for STR regions.
The tool uses the dynamic programming algorithm for alignment and
takes into account the STR repeat pattern, allowing size change in the
STR regions. In Kojima’s study, it performed better than ReviSTER and
the GATK IndelRealigner.
RepeatSeq [9] is an STR extraction tool that contains a realignment
procedure. It performs realignment after first mapping reads to a re-
ference and then uses a Bayesian statistical model to estimate the most
probable alleles based on the reference. In a comparison with the intial
lobSTR analyses, RepeatSeq was found to be able to assign genotypes to
repeats at a much higher rate, but did not make calls for some of the loci
called by lobSTR, most likely due to the disparity in the numerical re-
quirements for allele calling and the superior ability of lobSTR to detect
sequences that vary greatly from the reference.
STRviper [111] and CoalescentSTR [112] are also concerned with
repeat number estimation. Both implement novel statistical models for
repeat number estimation based on the difference between the actual
insert size and the insert size inferred from aligned, pair-end reads.
STRviper has been shown to outperform its predecessors, lobSTR and
RepeatSeq [111], but was later outperformed by CoalescentSTR [112].
The creators of lobSTR later developed a new tool, haplotype in-
ference and phasing for STRs (HipSTR)[113]. Designed for Illumina
sequence data, HipSTR learns the stutter characteristics of each STR
and uses this information to realign STR-containing reads and mitigate
the effects of PCR stutter. In a benchmark for genotyping accuracy,
HipSTR outperformed RepeatSeq and its predecessor, lobSTR.
4.2.2. STR detection by flanking sequence
A computational difficulty with the alignment approach to STR
detection is the mapping of reads to multiple locations, also called
multi-reads. In other words, sequence similarity between repeat motifs
of different STRs may result in the same read mapping to 2 different
loci. As the similarity between two repeat sequences increases, the
confidence in any read placement within the repeat decreases [50].
Since the source DNA is virtually never identical to the reference, the
alignment tool may map a read to a location it deems most similar to
the reference based on given parameters, rather than the correct loca-
tion.
The sequence-search approach tools were developed in response to
the limitations of pipelines that identified STRs only by sequence,
which are confounded by STR repeat complexity, novel alleles, and
sequence variation. Instead of finding STRs by alignment to a reference
by sequence or feature, sequence-search tools find STRs by the repeat
region flanks, which bypasses the difficulties that arise from STR se-
quence variation and complexity.
STR extraction tools for sequence-search pipelines are characterized
by the use of a STR information database and a string matching algo-
rithm. The data does not need to be aligned to a reference sequence.
The STR database contains the flanking sequences for each STR
locus and sometimes sequences of the STRs themselves. Such databases
are usually able to be curated by the user, allowing for the tweaking and
addition of sequences of interest. The information in the database is
used by the string matching algorithm, which scans through the data
file to find matching sequences. Usually, an approximate string
matching algorithm is used so that a degree of mismatch is allowed to
account for sequence variation. Upon finding a match, the read is
copied or extracted from the data file and collected as reads for the
allele.
The STRait Razor tool uses a plain-text database containing flanking
sequences, a short substring of the STR repeat motif and a dictionary of
allele lengths for each STR locus [100]. Up to version v2s [101,114], an
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Forensic Science International: Genetics 33 (2018) 117–128
approximate match program TRE-AGREP was used to find and extract
reads containing the flanking sequences. In version 3.0, a method of
accomplishing approximate matching using an exact match algorithm
with a prefix tree replaces TRE-AGREP for substring search [15].
The flanks are then trimmed off the STRs and reads not containing
the short substring of the repeat motif are filtered. The alleles are
named by sequence length and converted into the numerical allele
name using the allele length dictionary. By defining alleles by length
only, its capture of STR sequences is unaffected by sequence variation.
In Version 2s [101], configuration files were added for all com-
mercially available MPS multiplexes including SNPs, and also a data-
base for the conversion of STR sequences to recommended nomen-
clature, and a tool for visualizing the output data.
It is notable that the algorithm remained mostly unchanged up to
version 2s, whereas a new algorithm was used in version 3.0. We found
version 3.0 dramatically faster, typically taking a few seconds to com-
plete processing of a test FASTQ file, whereas v2s took a couple of
minutes. At stock settings, version 3.0 extracted a marginally lower
number of reads per locus.
STRait Razor has been used for multiplex system evaluation [5],
sequence variation [95], in a pipeline automated with Bpipe by Get-
tings et al. [115] and flank analyses [96].
MyFlq [14] also extracts STRs from FASTQ files. The tool maintains
a MySQL database of known alleles which is used to calculate a con-
sensus left and right flank for the STR locus. Reads are assigned to a
locus by the presence of PCR flanks, flanks are then removed and the
sequence is compared with the reference alleles in the allele database.
Based on the degree of the match, the reads are assigned flags to assist
manual interpretation of the alleles. Homopolymer stretches of the se-
quence are ‘compressed’ into a base during matching if no initial match
is found in the database. It is also available as an application with a
graphical user interface [116].
Other sequence-searching examples include STRinNGS and SEQ
Mapper. STRinNGS [16] takes FASTQ or unaligned BAM files as input,
and identifies STRs by flanking sequences. In addition to the STR se-
quence, STRinNGS also analyse flanking region variations by aligning
against hg19. SEQ Mapper [103] utilizes an allele library like MyFLq
and runs multiple searches for the presence of STR primers, flanks, and
STRs in different combinations. The multiple searches yield useful di-
agnostic information that may lead to the discovery of flank or se-
quence variations.
The dilemma with sequence-search strategies lies mainly in the
thresholds used for the approximate match algorithm and the varia-
bility present in the sequences. Setting a low, hard threshold may cause
more variable flanks to be missed while raising the threshold and al-
lowing more substitution and insertion/deletions may lead to false-
positive flank matches. Furthermore, if the flanks are insufficiently
different from another there is a potential for ambiguous matches.
When multiple flanks, genuine or otherwise, are found on one se-
quence, this may cause the extraction of multiple alleles from one read.
TSSV [102] functions in a similar manner to STRait Razor [100], but
instead of an approximate match algorithm, it uses semi-global pairwise
alignment to find STR flanks in reads. STRs are detected by the align-
ment of STR loci flanks onto a read and the set of flanks with the highest
alignment score (most similar to the aligned region) denote the locus.
Once a flank pair is aligned, the STR in between is extracted and
classified as a known or new allele and reported in a text output file.
By using alignment, a flank only locates to one position on the read,
circumventing the multiple flanks problem. However, the best scoring
flank alignment is not guaranteed to be the correct STR flank region and
STRs missing flanking region sequences are unlikely to be detected; a
comparison with lobSTR (publication version) revealed that TSSV is
better for recognizing complex repeats, while lobSTR is better for par-
tial reads [102].
Most of the above tools are concerned with the extraction of the STR
alleles, leaving the separation of minor alleles from stutter artefacts to
Y.-Y. Liu, S. Harbison
the analyst. FDStools is a software solution taking a stutter modelling
approach to identify stutter sequences [13]. In the tool, a database of
reference samples is consulted to generate a model for stutter, which is
applied to the alleles for profile correction.
4.3. Statistical analyses and databases
STRbase, an abbreviation of Short Tandem Repeat DNA Internet
DataBase, is a major online STR database [83]. It contains a collection
of STRs and allele sequences, a variety of DNA marker information,
training materials, and software tools. STRbase STR information has
been referenced in the creation of custom STR reference files for
alignment [2] and tools [14,16,100], as well as primer design [117].
STRidER [40] is an online allele frequency population database
based on allele numbers which could be queried or downloaded.
The STR Sequencing Project (STRSeq)[41] is a collaborative effort
between several laboratories to facilitate the sequence description of
STR alleles. The project has produced a catalogue of STR sequence di-
versity, guidelines on STR nomenclature, and STR sequences available
online as GenBank records.
5. mtDNA
5.1. Current state
mtDNA can be used to reveal lineage and is useful in situations
when nuclear DNA is unavailable, highly degraded, or where additional
lineage information is required [19].
Traditionally, mtDNA was analysed using Sanger sequencing
[118–121], focusing on the noncoding control region (CR), thought to
contain more sequence variation due to a higher mutation rate. The
analysis trend has moved from the specific regions of variation, hy-
pervariable segments I and II (HVS-I & HVS-II) and the hypervariable
segment III (HVS-III) to the entire control region [122,123] and sub-
sequently the whole mtDNA genome [124].
The move from the analyses of the hypervariable regions alone to
the whole mtDNA genome was facilitated by attempts to reduce ana-
lysis errors. Errors such as ‘artificial recombination’ caused by the
mixing of independently-amplified hypervariable segments from dif-
ferent individuals, low-quality raw data leading to sequence inter-
pretation mistakes and clerical errors led to the development of
guidelines to ensure that high-quality sequence was generated using
Sanger sequencing [122,125]. The discovery of useful polymorphisms
outside the HVS (and control region), and the availability of MPS
platforms, which offer much higher production of information at lower
costs than traditional Sanger methods, makes MPS a more suitable
option.
The ‘artificial recombination’ problem was ameliorated by the se-
quencing of the entire CR as one large amplicon, preventing mixture of
HVS segments. This led to the development of methods for targeted
MPS of the control region [126]. SNaPshot assays for population-spe-
cific SNPs in the control region have also been developed for population
screening [127] and are well-suited for degraded samples. A midi-sized
enrichment technique used for the CR was later applied to the enrich-
ment of the entire mtGenome [128].
Sequencing of the whole mtGenome revealed that sequence varia-
tions can exist in the mtDNA when the derived CR haplotypes are
identical [129,130]. These SNPs outside the hypervariable regions
provide additional resolution for mtDNA analysis.
Whole mtDNA sequencing allows analysis at maximum resolution
and recent studies using the MPS approach have successfully generated
haplotypes from blood [124,131] and single hair shaft samples [128].
Heteroplasmy, the presence of more than one mtDNA haplotype at a
nucleotide position, can be detected at lower levels using MPS due to its
high output [132,133]. The increased ability to assess low-level/minor
components also allows the study of the impact of DNA damage on the
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Forensic Science International: Genetics 33 (2018) 117–128
detection of heteroplasmic variations [45].
5.2. Workflows and pipelines
According to the guidelines for mitochondrial DNA typing set in
2014 by the International Society for Forensic Genetics, consensus se-
quences should be determined using dedicated software to align raw
reads to the rCRS. Variants should be recorded as the differences
compared to the rCRS, and confirmed by a second independent analysis
of raw data [125]. mtDNA data analysis traditionally uses the align-
ment approach. The reads are aligned to the rCRS using short read
aligners, then the base positions differing from the rCRS are extracted
from the alignment as variants, into a VCF file.
The VCF file contains the SNP variations required for the calculation
of a haplotype in a format supported by many downstream analyses
tools. MitoSAVE is one excel workbook-based tool that carries out such
haplotype conversion. Taking a VCF file as input, it produces a mtDNA
haplotype that follows standardized forensic conventions [134]. This
removes the need for manual translation of a VCF output file into a
haplotype, thus reducing clerical error.
Instead of manually running the tools for data processing, one could
use an automated pipeline. MToolBox is an example of such: it takes
BAM, SAM and FASTQ files as input and outputs a VCF file and hap-
logroup assignments [135]. It contains a mapper/remapper that aligns
reads to either the rCRS or RSRS, the GATK indel realigner, a variant
caller for generation of VCF, and a classifier for haplogroup assignment.
Analysis workflows that have been polished and packaged are of-
fered as commercial analysis software. Commercial software, such as
NextGENe and CLC Genomics Workbench, contain tools capable of
quality filtering, alignment and generation of mutation reports/variant
annotation and have seen frequent use in mtDNA studies [45,136,137].
AQME [17] is a toolkit for use in the user-defined mtDNA analysis pi-
peline within the CLC Genomics Workbench to further facilitate the
automated mtDNA analysis of sequence data and the production of a
forensic profile. GeneMarker HTS is a specialized mtDNA analysis
software designed for MPS/NGS data [138].
Sequencer onboard software is another example of a pre-packaged
pipeline. MiSeq reporter is the onboard software of the MiSeq se-
quencer. It uses the BWA aligner [139] for whole-genome alignment to
the rCRS, then the GATK [54] for variant calling. Variants obtained
then undergo reassignment to annotate areas of length heteroplasmy,
and repeats [124,134,140]. Data could also be downloaded from the
MiSeq Reporter software to be processed by alternative means [137].
On the Ion Torrent the variant caller plugin, which contains the
aligner TMAP (Torrent Mapping Alignment Program), generates a VCF
file from the sequence data stored on the server. The Ion Torrent soft-
ware has also been used in studies [136,141].
5.3. Databases
Haplogroup determination follows the acquisition of SNPs. A
number of mtDNA databases and tools are available online to facilitate
the process.
Phylotree is a regularly updated online phylogenetic tree of the all
known mtDNA haplogroups [142], currently comprising over 5400
haplogroups (Build 17). Displayed on a web page, the SNP mutations
are shown at each haplogroup branch. For most terminal haplogroups,
GenBank mtDNA sequences are provided for reference. Phylotree is a
tree diagram and does not provide any query functions, users find a
haplotype by tracing a path through the branching SNPs.
HaploGrep is an online mtDNA haplogroup classifier that uses
phylogenetic information from PhyloTree to determine the haplogroup
of mtDNA profiles [143,144]. File formats such as VCF and FASTA are
accepted as input and the haplotype is automatically calculated. Hap-
loGrep 2 produces a lineage diagram of the mtDNA haplotype showing
the mutations leading to the particular type. There are also tests that
Y.-Y. Liu, S. Harbison
can be run within the framework to detect recombination, phantom-
mutations and haplogroup discordance.
There are many other online mtDNA haplogroup classifiers that are
based on Phylotree. MitoTool [145] is available both as an online query
system and as a downloadable tool; it supports queries in sequences or
variants and generates a haplotype by alignment against rCRS and
RSRS. The tool accepts FASTA files containing the mtDNA sequence as
input and allows queries of specific regions such as the control region,
or the entire mtGenome. HmtDB [146] is a database containing human
mtDNA annotated with population and variability data generated with
the MToolBox tool [135]. The database offers queries in various criteria
such as geographical origin, haplogroups, haplotype-defining SNPs,
subject age group, and tissue type. mtDNAmanager [147] offers a web
interface to analyse quality, query haplotypes and store human mtDNA
sequences. It is limited to the control region sequences. A list of more
mtDNA tools is available online (https://isogg.org/wiki/MtDNA_tools).
The majority of haplogrouping tools base assignments on hap-
logroup-defining mutations, ignoring the extra information existing in
the form of private mutations. The sole reliance and focus on the
haplogroup-defining mutations without considering other distinctive
information may lead to incorrect haplogroup assignment. EMMA
[148] was designed to take private mutations into account during
haplotype estimation. Two databases are used for the estimation: one
comprised of full mtGenome sequences obtained from GenBank and the
other being the virtual haplotypes, each with haplogroup-defining
mutations, derived from Phylotree. Phylotree was used to draft haplo-
type estimates, while the whole mtGenome sequences were used to
calculate mutational stability that is applied to the haplotyping calcu-
lations.
EMPOP [38] is a forensic mtDNA database that holds high-quality
and directly accessible data. EMPOP is the main hub for mtDNA ana-
lyses and provides a number of tools for analysis and interpretation. A
string search algorithm, SAM, was devised for EMPOP database queries
that does not suffer bias when the query and database entry are an-
notated differently [149]. The query function is provided for finding
mtDNA sequences within the database by SNPs. The matches are shown
by origin and metapopulation with frequency values and graphed on an
interactive world map. The population function allows users to search
for published population data by fields such as accession number,
geographic affiliation, metapopulation and author. EMPOP does not
currently support input in VCF or FASTA format.
EMPOP also hosts three mtDNA analysis and interpretation tools:
the haplogroup browser, EMPcheck, and NETWORK (http://empop.
online/tools). The haplogroup browser (http://empop.online/hg_tree_
browser) is a searchable database of Phylotree haplotypes populated
with the mtDNA sequences from EMPOP by EMMA [148]. A hap-
logroup or haplotype is chosen and on an interactive world map, the
locations of where it occurs are graphed. Haplogroup-defining SNPs
could also be entered individually to find the haplogroup.
The EMPcheck tool (http://empop.online/empcheck) is used for the
validation of the EMPOP format file (.emp) to ensure that the standards
are met before submission of data to the database.
The NETWORK tool (http://empop.online/network) is used to ex-
amine the quality of mtDNA data by drawing a quasi-median (QM)
network, a web-like representation of the haplotypes that branch out
from a root haplotype-based on mutation events, with virtual haplo-
types (quasi-medians) joining the haplotypes. The QM network can be
used to confirm mutations or discover phantom mutations by con-
necting two samples and examining the shape of the graph. The QM are
the primary points of interest, and appearance of complex reticulations
due to a QM may be an error which should be confirmed by checking
the raw data.
6. Discussion and conclusions
As each of the three marker types, mtDNA, STRs, and SNPs may
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Forensic Science International: Genetics 33 (2018) 117–128
contribute valuable information to a forensic case, laboratories may
wish to have the bioinformatic facility to examine all three in a routine
manner. MPS, now affordable, is a compelling platform that can be used
to analyse all three of these markers. MPS offers higher-throughput, a
larger amount of data over Sanger sequencing methods and the se-
quence data generated is more informative than length data generated
by capillary electrophoresis.
This paper provides an overview of the processing of raw sequence
data into file formats accepted by a variety of analysis tools, common
workflows and tools, and the relevant databases that aid in inter-
pretation.
Whole mtDNA is currently analysed post-alignment, while STRs and
SNPs could be analysed with, or without alignment. As a result, pipe-
lines and tools for the unified processing of SNPs and STRs have begun
to emerge [14,101].
Many tools have been introduced, especially for STRs. Although
each tool was designed with theoretical strengths and weaknesses, it is
difficult to make a declarative statement that one tool or pipeline
functions better than another without a quantitative and qualitative
comparison of their output. A future study on the comprehensive
comparison between the available tools would steer the forensic com-
munity towards the optimal software solution.
As sequencers make base-calling errors, databases and population
data have been made useful in the conception of statistical values that
could be used for error correction. Currently, mtDNA analysis appears
to be the most well-established of the three markers in terms of analysis
protocols and databases. STRs as an MPS marker still require further
work in setting new nomenclature standards and repeat definitions.
In conclusion, many bioinformatics tools for forensic analyses exist
for the analyses of each marker system. As many as there are already,
tools and algorithms are constantly being developed in search of lower
processing times and novel algorithm paradigms.
Acknowledgements
This work was supported by the ESR Strategic Science Investment
Fund. We would like to thank Lisa Melia and Bethany Forsythe for their
helpful comments in reviewing this paper. This work was conducted by
Yao-Yuan Liu as part of the requirements for a Doctor of Philosophy
degree for The University of Auckland, New Zealand.
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