Exploring six NGS techniques that are transforming criminal investigations through advanced DNA analysis
When we think of forensic science, many of us picture the dramatic scenes from television crime shows where a single hair or tiny bloodstrap cracks the case wide open. While these depictions are often exaggerated, they reflect a fundamental truth: biological evidence is one of the most powerful tools in forensic investigation.
NGS enables extraction of wealth of information from crime scene samples, revealing ancestry, physical characteristics, and environmental history.
NGS successfully generates usable data from compromised materials that would be considered useless with traditional methods.
For decades, forensic scientists have relied on genetic techniques that look at just a handful of DNA markers. But what happens when evidence is too degraded, too complex, or too limited for these traditional methods? This is where Next-Generation Sequencing (NGS) is rewriting the rules of forensic genetics .
Imagine being able to extract not just a genetic fingerprint from a crime scene sample, but a wealth of information about the person who left it—their ancestry, physical characteristics, and even the type of environment they recently inhabited. NGS makes this possible by reading DNA sequences on a massive scale, simultaneously analyzing millions of DNA fragments to reveal information that was previously inaccessible 2 .
At its core, Next-Generation Sequencing is a high-throughput technology that determines the order of nucleotides in DNA or RNA molecules. Unlike traditional Sanger sequencing, which processes a single DNA fragment at a time, NGS works through massively parallel sequencing, simultaneously reading millions to billions of DNA fragments 2 4 9 .
DNA is fragmented into smaller pieces and attached to adapters—short, known DNA sequences that allow the fragments to be handled in subsequent steps 6 7 .
Each DNA fragment is amplified (copied multiple times) to create clusters of identical fragments, strengthening the signal for detection during sequencing 6 .
Using a method called "sequencing by synthesis," specialized enzymes build complementary strands on the DNA templates while a camera detects which nucleotide is added at each step 2 6 .
Advanced bioinformatics tools piece together the short sequenced fragments by aligning them to reference genomes, identifying variations that might be forensically relevant 6 .
NGS can generate results from incredibly small amounts of DNA—crucial when crime scene evidence is limited .
Instead of performing separate tests for different types of markers, NGS can simultaneously analyze autosomal DNA, mitochondrial DNA, Y chromosomes, and other markers in a single test .
Beyond just identification, NGS can reveal phenotypic information such as hair and eye color, biogeographical ancestry, and age-related markers 5 .
The ability to sequence short DNA fragments makes NGS particularly valuable for analyzing degraded evidence that would fail with conventional methods .
| NGS Technique | Primary Forensic Application | Key Advantage | Limitation |
|---|---|---|---|
| Whole Genome Sequencing | Comprehensive profile generation, difficult cases | Unbiased coverage of entire genome | Higher cost, more complex data analysis |
| Targeted Panel Sequencing | Specific marker analysis (STRs, SNPs) | Cost-effective for focused questions | Limited to pre-selected regions |
| Mitochondrial DNA Sequencing | Maternal lineage, degraded samples | High copy number per cell | Limited discriminatory power alone |
| RNA Sequencing | Body fluid identification, time since deposition | Identifies tissue sources | RNA more labile than DNA |
| Epigenetic Sequencing | Age estimation, differentiating identical twins | Reveals gene regulation patterns | Emerging technology, complex interpretation |
| Microbiome Sequencing | Crime scene characterization, geolocation | Environmental context | Variable microbial communities |
Collection of biological material must be performed with meticulous care to avoid contamination.
Extraction of DNA/RNA requires specialized methods that maximize yield, purity, and quality.
DNA is fragmented and adapters are ligated to the ends of DNA fragments.
Massively parallel sequencing occurs on specialized platforms using sequencing by synthesis.
Bioinformatics pipelines process raw data through demultiplexing, alignment, and variant calling.
Genetic variations are translated into forensically relevant information for legal proceedings.
The forensic NGS process begins where any investigation does—at the crime scene. Collection of biological material must be performed with meticulous care to avoid contamination. Unlike research samples, forensic evidence is often limited, degraded, or mixed with inhibitors that can complicate analysis 6 .
The isolation of nucleic acids (DNA or RNA) requires specialized methods that maximize yield, purity, and quality even from challenging samples like bones, teeth, or formalin-fixed paraffin-embedded (FFPE) tissues. The extracted DNA must be free of compounds that could inhibit enzymes used in later steps of the process. For particularly degraded samples, whole genome amplification may be employed to increase the amount of available template 6 .
Library preparation is perhaps the most crucial step in forensic NGS, as it determines what sequences will be available for analysis. Two main approaches are used:
DNA is physically sheared into smaller fragments, ideal for various DNA input types and minimizing sequence artifacts 7 .
Uses enzymes to cut DNA, producing tunable and reproducible fragment sizes—better for automated, high-throughput processing 7 .
During library preparation, adapters are ligated to the ends of DNA fragments. These adapters serve as handling elements, allowing the fragments to be captured on sequencing flow cells and recognized by sequencing reagents. For forensic applications, unique molecular identifiers are often incorporated to track individual samples and prevent cross-contamination 6 7 .
To understand the real-world value of NGS in forensics, consider a simulated study designed to test how different sequencing methods perform with compromised samples typical of crime scene evidence. Researchers created samples with various challenges: some were chemically degraded to mimic environmental exposure, others were mixed with multiple contributors as often happens in real evidence, and some contained extremely low quantities of DNA .
The findings from such experiments demonstrate why NGS is becoming increasingly important in forensic genetics:
While traditional methods failed to produce complete profiles from significantly degraded samples, NGS successfully generated usable data from even the most compromised material. This is because NGS can work with shorter DNA fragments that remain when samples degrade .
In samples containing DNA from multiple contributors, NGS provided better resolution of individual contributors, particularly when combined with single nucleotide polymorphism (SNP) analysis that offers more genetic markers for separation.
NGS methods successfully generated profiles from samples with as little as 100 picograms of DNA, far below the requirements of conventional techniques.
| Sample Type | Traditional Capillary Electrophoresis | Targeted NGS Panels | Whole Genome NGS |
|---|---|---|---|
| Highly Degraded DNA | Partial profile (8-12 of 20 markers) | Nearly complete profile (90% markers) | Complete genomic data but with coverage gaps |
| 4-Person Mixture | Limited contributor separation | 3 contributors identified | All 4 contributors identified |
| Low Template (100 pg) | Unreliable or no results | 75% markers detected | 20x coverage across 70% of genome |
| Inhibitor-Present | Failed analysis | Successful with adjusted protocol | Successful with adjusted protocol |
The implications of these results are profound for forensic practice. NGS technologies enable analysis of evidence that would previously have been considered unsuitable for genetic testing—whether because of degradation, minute quantity, or complex mixtures. This expands the range of cases that can be solved through DNA evidence and enhances the statistical weight that can be assigned to matches when evidence is suboptimal.
Implementing NGS in a forensic laboratory requires not just sequencing instruments but also a suite of specialized reagents and materials. These components have been optimized to handle the unique challenges of forensic evidence.
| Reagent/Material | Function | Forensic Specific Considerations |
|---|---|---|
| Nucleic Acid Extraction Kits | Isolate DNA/RNA from various sample types | Optimized for challenging samples (bones, teeth, FFPE) |
| Library Preparation Kits | Prepare DNA for sequencing | Designed for low-input, degraded DNA; include unique indexes for sample tracking |
| Enzymatic Fragmentation Mix | Cut DNA into sequenceable fragments | Controlled fragment size important for degraded samples |
| Mechanical Fragmentation Systems | Physically shear DNA | Alternative to enzymatic methods; reduces bias |
| Adapter Ligands | Attach sequences for platform recognition | Often include molecular barcodes to identify individual samples |
| Target Enrichment Panels | Capture specific genomic regions of interest | Include forensic-relevant markers (STRs, SNPs, mitochondrial DNA) |
| Sequencing Chemistry | Enable nucleotide-by-nucleotide reading | Balanced for read length, accuracy, and output |
| Bioinformatic Software | Analyze raw sequence data | Specialized tools for forensic interpretation, mixture analysis |
The selection of appropriate reagents is critical for forensic success. For instance, library preparation kits specifically designed for low-input DNA ensure that precious evidence isn't wasted in the sequencing process. Similarly, target enrichment panels that focus on forensically informative markers maximize the value of each sequencing run for identification purposes 3 7 .
Forensic laboratories must also implement rigorous quality control measures at each step of the NGS workflow. This includes validation of reagent performance, monitoring of sequencing run metrics, and verification of bioinformatic pipeline accuracy. These controls are essential not just for scientific reliability but also for ensuring that results will be admissible in legal proceedings.
The integration of NGS into forensic science continues to evolve, with several promising developments on the horizon.
Microbiome analysis represents an exciting frontier, where the bacterial communities associated with human samples might provide clues about a person's recent geographical movements, lifestyle habits, or even the time since deposition of biological evidence .
The miniaturization of sequencing technology is another promising direction. Portable sequencing devices that can be deployed in field investigations could revolutionize crime scene processing, allowing for rapid preliminary analysis without the delay of laboratory processing.
As these technological advances proceed, they must be accompanied by appropriate ethical frameworks and legal guidelines. The ability to extract increasingly detailed personal information from minute biological samples raises important privacy concerns that society must address.
The forensic science community needs to establish standardized protocols and interpretation guidelines to ensure that NGS-based evidence is reliable and reproducible across different laboratories.
Next-Generation Sequencing represents more than just a technical upgrade for forensic science—it fundamentally expands our ability to extract information from biological evidence. By enabling comprehensive genetic analysis from samples that previously would have been considered inadequate, NGS is opening new avenues for solving crimes and identifying victims.
The six NGS techniques we've explored—from targeted panels for specific markers to whole genome sequencing for comprehensive analysis—each offer unique advantages for different forensic scenarios. While challenges remain in standardization, interpretation, and integration into legal frameworks, the potential of these technologies to enhance justice is undeniable.