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Retrotransposable elements (REs) consisting of long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) are a group of markers that can be useful for human identity as well as bio-ancestry testing. SINEs are a class of REs that are typically less than 500 nucleotides long; while LINEs are typically greater than 500 nucleotides and up to several thousand base pair long. The third type of RE is the composite retrotransposon known as SVA (SINE/VNTR/Alu) elements.
There are several advantages of RE’s for the application of human identity and bio-ancestry testing. First, the bi-allelic nature of RE's provides technical simplicity of typing two alleles per genetic marker instead of typing an array of nucleotides, sometimes hundreds of nucleotides in length, as with the conventionally used STRs. Secondly, RE's are identical by descent only, making them more stable than other systems in terms of inheritance. In addition, REs do not yield stutter artifacts due to slippage during the PCR, which can reduce some interpretation issues associated with the currently utilized STRs. Until now, however, due to the inherent size difference (>300 bp) associated with insertion and null alleles (or INNULs), the use of REs has not been practical for human identity applications. Although the use of SINEs such as Alu in determining human identity has been studied and reported in the literature, the more than approximately 300 bp size difference between the two alleles prevented development of any useful system. This is the first time any research on the use of LINEs, SINEs, or SVA element insertions, as a multiplexed system, has been proposed.
To circumvent the allele size disparity, we have developed a novel primer design methodology that essentially removes the intra-specific locus competition that occurs in heterozygotes. This involves utilization of the direct repeat units that flank an Alu element. The novel primer design reduces overall amplicon size among loci as well as the difference in amplicon size between the two allelic states of INNULs. The resulting INNUL allelic amplicons can be designed to differ by as little as one base pair instead of the approximately 300 bp Alu insertion. Additionally, the amplicon size has been reduced substantially, to a size much smaller than currently used STR markers, such that highly degraded DNA samples can successfully be profiled with nuclear DNA. Utilizing this primer design, a more simplified, rapid and automated technology can be applied to the analysis of LINE, SINE and SVA insertion polymorphisms [
The novel primer design enables these markers to be applied to human identity testing in several ways, including their use in a typing system fully compatible with current capillary electrophoresis platforms, in a real time qPCR assay for quality and quantity assessment, in Rapid DNA analysis platforms, and in Next Generation Sequencing (NGS) platforms. A typing system consisting of 20 RE’s and Amelogenin in a single multiplexed amplification typing system, InnoTyper®21, has been developed. The novel primer design was successful in significantly reducing the size of the amplicons for all 21 markers to between 60 bp and 124 bp. The small amplicon sizes result in an extremely sensitive, rapid and useful multiplex for highly degraded forensic samples. When evaluating 3 major North American populations, these markers generally met Hardy-Weinberg expectations and showed little evidence of detectable levels of linkage disequilibrium between the markers tested. Data supports the usefulness of this system in analyzing forensic DNA samples with high discrimination power for samples that did not produce usable STR results, including rootless hair shafts and severely degraded human remains. Fig. 1 shows InnoTyper® 21 results from a rootless 2 cm hair shaft. This system is therefore expected to provide strong utility for analyzing single source degraded DNA samples such as those found in mass disasters as well as other human identification efforts. The bi-allelic nature of this Alu typing system helps to simplify analysis but does present interpretation challenges for samples containing a mixture of more than one DNA donor, when using current capillary electrophoresis platforms. However, these markers are applicable to mixture analyses if used on an NGS platform. With NGS, hundreds of markers can be sequenced in a massively parallel fashion, yielding substantially more throughput and minimizing the need for the fragment-cloning methods that are often used in Sanger sequencing.
A second application for these markers in human identity is the development of quantity and quality assessment real time qPCR systems known as InnoQuant® [
] and InnoQuant® HY. These next generation DNA quantification kits allow accurate quantitation at picogram levels of two autosomal targets: a “short” RE target of 80 bp in size, and a “long” target (from a separate RE) of 207 bp in size. The large copy number of the selected targets (>1000 copies/genome) provides high sensitivity for low level samples while minimizing the effect of variation between individuals thus enabling high reproducibility. The InnoQuant® HY kit also includes multi-copy targets on the Y chromosome which enable the detection of male DNA at picogram levels. Sensitivity of the qPCR InnoQuant® HY kit demonstrates an average fold change from the expected values of 8%, 6%, and 8% for the long, short, and Y targets, respectively, in a serial dilution of genomic DNA in expected quantities of 100 ng/μL to 0.0006 ng/μL. The 0.6 picograms per microliter (approximately a tenth of a cell) data point shows minimal fold changes from the expected quantities with the InnoQuant® HY kit. Even at sub-picogram levels (i.e., the 0.0006 ng/μL data point), this kit shows an average difference from the expected quantities of 19% across all 3 targets. InnoQuant® and InnoQuant® HY enable confident screening of negative samples and can guide selection of optimal downstream typing methods and input DNA target amount, based on the sample's quantitation and extent of degradation.
Yet a third application of these InnoTyper® 21markers is their combination with ancestry informative ALU (AI-ALU) markers, all small amplicon sizes of less than 100 bp, to provide additional identity information. ALU REs are exceptional markers for tracking identity by descent and inferring biogeographic origin due to their high copy number, known ancestral state, and genomic stability. We have selected 30 AI-ALUs most informative for bio-ancestral identification from the ALUs identified and curated by the 1000 Genome Project Structural Variations Group. These AI-ALUs were selected from a data set of 2504 unrelated individuals from 26 populations and therefore provide significant resolution of biogeographical origin.
InnoTyper® RE markers can be combined with ancestry informative ALU RE markers, all of small amplicon size < 100 bp, to provide additional information from challenging forensic samples. Such an NGS-based targeted-capture multiplex panel can include ∼100 small amplicon RE markers to provide increased discrimination power for human identity purposes, determination of ancestry within continental and sub-continental population groups, accurate differentiation of complex mixtures, and effective typing of poor quality (i.e., highly degraded, low-level) samples. The development of a small amplicon, multiplexed primer kit for preparing next-generation sequencing (NGS) libraries will prove extremely useful for forensic and bio-ancestral identification from challenging DNA samples.
In summary, RE's are novel markers that, due to their high copy number in the genome, can provide the most sensitive and reproducible DNA quantification and quality assessment systems available for forensic applications. In combination with the novel primer design described above, a multiplexed InnoTyper®21 system of small amplicons has been developed for standard PCR/CE platforms that is highly sensitive and can be utilized for human identification purposes with severely degraded and low quantity forensic samples. The RE markers will also serve as exceptionally informative bio-ancestry markers providing additional information from challenging unknown evidence samples.
This material is based upon research supported by the National Science Foundation under SBIR Grant No. 1230352.
S.K. Sinha, et al., Oral presentation at the 66th Annual Meeting of the AAFS Seattle, WA.