Forensic DNA Analysis: CE or MPS?
Forensic DNA analysis utilizes autosomal short tandem repeat (STR) markers to profile DNA, establishing identity of missing persons, confirm kinship, or link individuals of interest to crime scenes. Capillary electrophoresis (CE) based Fragment Analysis was and still is the most widely used standard method in forensic DNA analysis. Its greatest strengths are the standardization of loci used by all laboratories and an extremely large searchable database of genetic profiles (e.g. Combined DNA Index System, CODIS), a relative fast turnaround time and perceived cost of analysis. However, CE has limitations and challenges, particularly when dealing with highly degraded or low quantity DNA samples and mixture deconvolution.
Next-generation Sequencing (NGS) technology, also called Massively Parallel Sequencing (MPS), provides significantly increased power of discrimination of STR alleles. Combined with the tremendous amount of information content from expanded sets of core STR loci and supplemental genetic markers (Single-Nucleotide Polymorphisms, or SNPs, and microhaplotypes), it enables the deeper deconvolution of complex mixed poor quality or low quantity forensic samples. Moreover, a single MPS run interrogates hundreds of forensically relevant genetic markers, eliminating the need to for using multiple fragment length‑based STR kits. Increased adoption of MPS by the forensic community in high-demanding investigations demonstrates that MPS has the potential to be the next standard for genetic analysis in routine forensic casework.
Transition from CE to MPS-based DNA Analysis
Forensic DNA analysis is at an important stage in its development. New technologies have accelerated forensic research applications, and society - legal and law enforcement in particular - has more than ever before come to realize just how beneficial forensic DNA analysis can be for their own purposes. Consequently, customers of forensic laboratories are becoming more demanding, which brings new challenges, as laboratories become increasingly hindered in their forensic casework:
- Sample throughtput, due to increasing number of (cold) cases and sample backlogs
- Sample quantity available (input)
- Sample quality, i.e. highly degraded samples, from a variety of sample sources and matrices
- Discrimination power relative to paternity and deconvolution of mixed DNA samples
- Technical and workflow limitations of current DNA-based methods used
- Result interpretation and conclusion (science versus law)
- Validation and certification of methods used
IDseek® comprises a complete range of STR, SNP and mitochondrial DNA (mtDNA) based Massively Parallel Sequencing (MPS) solutions for Forensic DNA analysis, based on Reverse Complement PCR (RC-PCR) powered library prep in combination with illumina® sequencing.
The IDseek® assay is the first MPS assay with a fast and straightforward targeted library prep workflow that can compete with capillary electrophoresis (CE) based assays for routine application in Forensic casework.
The IDseek® MPS Library Prep workflow provides multiplex targeted amplification, sequencing indexing and adaptor addition occuring simultaneously in a single closed tube reaction. After RC-PCR amplification, samples are pool-purified and size-selected using AmpliClean™ Magnetic Bead Cleanup, providing sequencing ready libraries.
MPS Library Prep Assays in Routine Forensic Casework
IDseek® MPS Library Prep Kits add significant benefits over classic CE analysis:
- RC-PCR simplicity and security: multiplex targeted amplification, sequencing indexing and adapter addition, simultaneously performed in a single closed tube
- Powerful RC-PCR kinetics: robust and sensitive assays, even on extremely low quality input, raw material, and complex mixed DNA traces
- Small amplicons facilitate analysis of highly degraded samples typically found in forensic casework
- Greater multiplexing of autosomal STRs, Y-STRs, SNPs, and mitochondrial DNA control regions, avoiding the need for separate, secondary workflows
- Full base-by-base sequencing in and around the repeat motifs
Library prep workflow automation is possible, allowing high-throughput analysis
- Automated data analysis
IDseek® MPS Library Prep kits have been tested with Tannic Acid, Humic Acid and Bacterial DNA. RC-PCR technology allows the reliable analysis of highly degraded samples frequently found in forensic casework.
With IDseek® MPS Library Prep all amplicons are designed to meet the shortest possible fragment lengths and are compatible with illumina® MiSeq™, NextSeq™ 1000 and MiSeq™ FGx sequencing systems, with 2x10 bp Unique Dual Index reads. The combination of state-of-theart features with the possibility for automated data analysis (using e.g. STRmix™ and MixtureACE™ DNA Analysis Software) without human intervention makes IDseek® MPS Library Prep kits the ideal solution for the transition from CE to MPS.
MiSeq and NextSeq are trademarks of Illumina. MiSeq FGx is a trademark of Verogen.
IDseek® OmniSTR™ MPS Full Concordance with Validated CE Techniques
“We really liked the OmniSTR™ kit. We particularly appreciated the straightforward sample preparation with short hands-on time compared to other MPS library preps. NimaGen was very helpful throughout the testing phase and supported us in data analysis. We observed full concordance with validated CE techniques and were positively suprised by the high sensitivity of the analysis.”
IDseek® MPS Library Prep for STR Profiling
IDseek® OmniSTR™ Global Autosomal STR Profiling Kit provides an RC-PCR powered MPS library prep solution for analysis of 31 autosomal STRs relative to establishing identity of missing persons, confirm familial relations, and link persons of interest to crime scenes. Covering all relevant STR core loci from USA, EU, UK, and Interpol, plus SE33, D4S2408, D6S1043, D9S1122, D17S1301, D20S482, PentaD, PentaE and DYS391.
The IDseek® mYSTR™ Kit is a single reaction, multiplex library prep kit for Y-chromosomal STR haplotyping. It ampliflies 27 Y-chromosomal STR markers plus Amelogenin, to characterise paternal lineages of unknown male trace donors, as the Y-chromosome is passed genetically from paternal grandfather to father to son. The analysis of haplotype (the set of STR alleles typed on a single Y chromosome) is an established method in forensic casework analysis, paternity testing and genealogy.
IDseek® MPS Library Prep for SNP Profiling
IDseek® OmniSNP™ Identity Informative SNP Typing Kit provides an RC-PCR powered MPS library prep solution for high-resolution profiling of 85 high-variable phenotypic human identity SNPs, to discern small differences both within a population and among different populations. Covering relevant, established SNP marker sets implemented by laboratories in forensics, human identity and paternity testing.
IDseek® Mitochondrial DNA Sequencing
IDseek® Mitochondrial HVR provides an RC-PCR powered MPS library prep solution for analysis of hypervariable regions (mutation hotspots) in very low quantities of (highly degraded) nuclear mtDNA. In complex samples that do not provide a full STR or where routine STR analysis of samples has failed, such as hair, bones and teeth. For laboratories in those countries where full genome sequencing in forensics is restricted by guidelines or regulations.
For analysis of hypervariable regions (mutation hotspots) with minimal DNA input of highly degraded nuclear mtDNA. For complex samples that do not provide a full STR, or where routine STR analysis of samples has failed. The Full Genome capability provides greater yields from small samples, and offers more critical information relative to identifying missing persons, disaster victims, population studies, or mitochondrial genomes from ancient remains relative to archaeological sites.
IDseek® MPS Library Cleanup and Size Selection
The AmpliClean™ Cleanup Kit provides highly efficient and cost-effective magnetic bead-based technology for high-throughput purification of PCR products. AmpliClean™ removes, salts, primers, primer-dimers and dNTPs, leaving ultra-pure DNA of the desired length. It requires no centrifugation or vacuum filtration steps. Purified PCR products can be used directly for Sanger cycle sequencing. The AmpliClean™ protocol is highly flexible for manual operation and can be easily incorporated in any liquid handling system for automated processing. AmpliClean™ magnetic beads are the widely adopted alternative to AMPure XP beads for purification.
AMPure XP is a trademark of Beckman Coulter.
High-quality NGS libraries are key to keeping NGS costs down, minimizing the risk of losing important genetic information, minimizing wasted sequencer capacity and maximizing usable data. Effective library cleanup, size selection, quantification and validation are pivotal in generating high-quality NGS results and reliable data.
More value can be added when MPS libraries are prepared in a convenient, safe and cost-effective fashion. Read more about the IDseek® pooled MPS library cleanup and size selection approach.
Leading, Expert DNA Analysis Tools
For DNA analysis, you want the right tools to help you generate quick, accurate results. NicheVision Forensics can help. NicheVision develops and distributes innovative DNA analysis software for forensic laboratories worldwide. Its next-generation expert softwares STRmix™ and MixtureAce™ are used by leading forensics laboratories around the world.
STRmix™ and MixtureAce™ DNA analysis tools will meet your needs – and help you find the answers you are looking for in routine and specialty forensic casework.
Generating accurate, high-resolution interpretations of the most complex DNA mixtures, helping DNA analysts interpret data derived from both Fragment Analysis and Massively Parallel Sequencing.