The Power of MPS in Forensic DNA Analysis

After more than thirty years of DNA analysis in forensics using classical methods such as PCR followed by Fragment Analysis by capillary electrophoresis (CE), new technologies based on Massive Parallel Sequencing (MPS) are now changing this field. In addition to unravelling complicated mixed DNA traces, it can also reveal far more information and be used with lower quality and highly degraded samples. Existing MPS library preparation methods are complicated, typically involving multiple hands-on steps, error-prone workflows, increased risk for amplicon contamination and sample swapping. With the introduction of the novel and innovative Reverse Complement PCR (RC-PCR) technology, NimaGen opens doors for the forensic community to make the transition from classic CE to MPS in the safest and simplest way possible, without compromise.

Reverse Complement PCR (RC-PCR) Technology

DNA Sequencing methods typically involve multiple steps, whether it is classical Sanger Sequencing or MPS. RC-PCR from NimaGen is a breakthrough technology providing MPS library prep in a single, closed-tube workflow, with extremely low handling. Multiplex targeted amplification, adapter addition and Indexing all occur simultaneously in a single, closed tube reaction: simply add your DNA to the pre-dispensed index tubes and run the PCR reaction in your thermal cycler. As a direct consequence of the Unique Dual Indexing in the very first step of the library prep (“Early Indexing”), and the single-step amplification, the risk of sample swapping, PCR contamination and pipetting errors are drastically minimized. Additionally, the reaction kinetics of RC-PCR results in high sensitivity and specificity, due to the fact specific primers are synthesized during the reaction, so concentrations of primers and amplicons are more in line. This reduces potential primer dimerization and off-target primer binding. MPS targeted library prep will never be easier. See video animation of the working principle at www.rcpcr.com.

IDseek® MPS library prep: How does it work?

NimaGen offers a range of forensic MPS library prep kits that all work with the same principle. Libraries from different kits can be combined in a single Illumina® run, to make efficient use of your sequencing capacity. Every kit consist of two parts. One assay specific part, that includes the target specific probe mixes and the RC-PCR Master Mix, and one universal part: The IDX plate(s), containing pre-spotted and dehydrated Unique Dual Index primers. All IDX plates are breakable per 8 wells, to maximize efficiency.

IDseek® Multiplex STR Kits for MPS

Short Tandem Repeat (STR) analysis is by far the most used tool in forensic DNA analysis for generating unique genetic profiles. Forensic science uses the variability in STR lengths within the human population to enable scientists to distinguish between different DNA samples and run them against databases to obtain a match. In the classic analyzing method, DNA fragments are separated and detected using capillary electrophoresis (CE) in order to determine allele length. MPS technologies add significant benefits over CE analysis, such as not being limited by the number of available fluorescent channels or primer design restrictions due to amplicon size or length constraints. Taken together, these benefits allow MPS to overcome a number of complicating factors:

• Allowing the recovery of fuller profiles from challenging samples due to reduced amplicon sizes
• Providing the full base sequence in and around the repeat motifs, further increasing the discriminative power of STR markers.
• Higher multiplexes for more STRs to be detected simultaneously avoiding the need for separate secondary workflows.
• Differentiation of genetic profiles from complex mixtures, commonly associated with crime scenes.

All amplicons in NimaGen’s STR multiplex kits are designed to meet the shortest possible fragment lengths and are compatible with Illumina® MiSeq™ systems, with 2 x 10 bp Unique Dual Index reads. This combination of state-of-the art features with the possibility for automated data analysis without human intervention makes this kit the ideal solution for the transition from CE to MPS.

IDseek®OmniSTR™ Global Autosomal STR Profiling

The OmniSTR™ Kit is a single reaction, multiplex library prep kit for autosomal STR profiling, including all the US and European expanded core loci, plus SE33, D4S2408, D6S1043, D9S1122, D17S1301, D20S482, PentaD, PentaE and DYS391. All targets have been designed for short amplicons, while maintaining the most informative sites in the flanking regions. Forensic STR profiling by MPS tend to be applied in specialty cases due to high hands-on time investments. The unmatched simplicity, robustness, sensitivity and safety of IDseek® now facilitates a routine MPS application, hardly exceeding hands-on time of CE.

IDseek® mYSTR™ Y-Chromosomal STR Profiling

The mYSTR™ kit is a single reaction, multiplex library prep kit for Y-Chromosomal STR profiling, amplifying 27 Y-chromosomal STR markers plus Amelogenin in a single multiplex. All targets have been designed for short amplicons, while maintaining the most informative sites in the flanking regions. Forensic STR profiling by MPS tend to be applied in specialty cases due to high hands-on time investments. The unmatched simplicity, robustness, sensitivity and safety of IDseek® now facilitates a routine MPS application, hardly exceeding hands-on time of CE.

IDseek® Mitochondrial DNA HVR and Full Genome

The use of MPS for the analysis of Mitochondrial DNA (mtDNA) adds great benefits for Forensic Scientists, especially for samples like hairs, teeth and bones, coming from crime scenes or disasters, or ancient DNA samples. Also, for the analysis of any samples that contain very low quantities or highly degraded nuclear DNA, mtDNA analysis can add extra power in creating genetic profiles. Since every human cell contains up to 2000 mitochondria, and the mtDNA is more stable than nuclear DNA, the higher sensitivity of mtDNA analysis can add additional tools to the standard autosomal or Y-chromosomal genetic profiling. With the introduction of the IDseek® mtDNA sequencing assays, the High Variable Region (HVR) or the entire mtDNA genomes can be easily sequenced, providing additional information, like haplotypes and heteroplasmy. Like every IDseek® assay, the workflow is extremely straightforward and safe. In contrast to the STR and SNP assays, the mtDNA kits have two probe pools per sample, separating overlapping fragments in order to obtain full length sequences after alignments. The IDseek® Mitochondrial DNA HVR Sequencing kit is based on two (2) multiplex reactions of 5 amplicons each. The amplicon lenghts are 150 – 230 bp. The IDseek® Mitochondrial DNA Full Genome Sequencing is based on two (2) multiplex reactions of 50 amplicons each. The amplicon lenghts are 150 – 250 bp.

IDseek® OmniSNP™ Identity Informative SNP Typing

New technologies have increased the spectrum of sample types that can be subjected to DNA analysis. While several human ID markers are offering amplification of short amplicons, several forensic samples (e.g., human remains and touch evidence) do not always contain sufficient quantity and quality of DNA to be typed with current methodologies. As a result, challenging samples with high DNA fragmentation may not contain sufficient intact templates for successful PCR amplification. Several studies have reported that DNA fragments in highly degraded samples are very short down to approximately 50 bases in length. Only a limited number of DNA markers, if any, may be detected with such short fragments. Several methods to improve DNA recovery from compromised samples have been developed but had limited or no success. Two advances now may make it possible to analyze highly degraded DNA: Reverse Complement PCR (RC-PCR) and MPS. With the design of the OmniSNP™ panel, fragments as short as approximately 50–100 nucleotides in length are targeted, increasing the success of typing targets from highly degraded DNA samples.

IDseek® comprises a complete range of STR, SNP and mtDNA based Massively Parallel Sequencing (MPS) solutions for Forensic DNA analysis. Come learn more about the Next Revolution in Forensic MPS here!

Reverse Complement PCR Features and Benefits:

Cost-efficient workflow

• Breakable index plates ensures optimal usage, minimizing waste
• One closed-tube, single reaction workflow with simultaneous indexing and target amplification reduces labor time
• Single tube sample pooling for library clean-up significantly reduces usage of required magnetic beads and consumables
• Well-balanced read distributions maximizes sequencing instrument flow cell capacity

Confidence in test results

• Closed-tube RC-PCR workflow significantly reduces hands-on time and pipetting error, minimizing risk of sample contamination minimizing risk of sample contamination
• Sample tracking dye in pre-spotted UDI plates ensures accuracy
• Unique RC-PCR kinetics promote high-target specificity and coverage uniformity (optimized read depth balance) from low DNA input