Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets

Loftus, Andrew F.; Murphy, Gina; Brown, Hiromi; Montgomery, Anne; Tabak, Jonathan; Baus, James; Carroll, Marion L.; Green, André; Sikka, Suresh C.; Sinha, Sudhir K.

doi:10.1016/j.fsigen.2017.04.009

Cited by 27 publications

(14 citation statements)

References 27 publications

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“…However, the assay is based on high‐resolution melting and not on hydrolysis probes, which provide higher specificity. Commercial kits have shown sensitivity around 1 pg/µL [4–6]. That is achievable because they target multi‐copy genomic loci and the sequence of the oligonucleotides is unknown to the public.…”

Section: Discussionmentioning

confidence: 99%

“…For over 10 years, multiplex qPCR for DNA quantitation has been reported in forensics [2] and it is considered the gold standard for human‐specific DNA quantitation. Nevertheless, the assays have been developed and validated on platforms that detect more than three different dyes [3–9]. With the spreading of qPCR technology, smaller, more economic and user‐friendly platforms have reached the market.…”

Section: Introductionmentioning

confidence: 99%

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DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

et al. 2020

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DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratoriesFor over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

et al. 2020

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“…Eleven of the 13 teeth samples were typed with both Identifiler Plus and ForenSeq DNA Signature (DPMB, 27-sample multiplex on MiSeq FGx) and resulted in concordant profiles 13 of the remaining teeth extracts were subsequently quantified with InnoQuant HY ( Table 3). Advantages that were observed of the InnoQuant HY system compared to NanoDrop include the following: human specific results, total human and total male concentrations provided, quality information with degradation indexes, and an IPC for degradation and inhibition detection [8,9]. InnoQuant HY quantification results indicated that root cement and dental pulp tissues recovered using the DFK method yielded DNA of sufficient quality and quantity for the majority of the teeth samples.…”

Section: Teeth Samplesmentioning

confidence: 99%

“…Quantity and quality of total human and total male DNA can be assessed using the InnoQuant® HY Kit (InnoGenomics Technologies) [8,9]. Based on real-time qPCR quantification of high copy number Alu and SVA retrotransposable elements (> 1000 copies per genome), this method assesses autosomal targets of two lengths (short and long) to enhance sensitivity and reproducibility of DNA quantitation and degradation detection in forensic samples [8,9]. Quality information includes detection of polymerase chain reaction (PCR) inhibitors and determination of a degradation index (DI), which correlates strongly to amplification and genotyping success [8,9].…”

Section: Introductionmentioning

confidence: 99%

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing

Carrasco

Inostroza

Didier³

et al. 2019

Int J Legal Med

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Human dental remains encountered in criminal casework evidence, missing person cases, or mass disaster tragedies provide a valuable sample source for DNA typing when suitable soft tissue is unavailable. Using traditional methods, teeth samples can be challenging to process, resulting in low-quantity and/or quality nuclear DNA and insufficient profiles for comparisons. This study examines the performance of a three-part nuclear DNA analysis workflow for teeth samples based on (1) improved dental tissue recovery using the Dental Forensic Kit (DFK MR) (Universidad de los Andes) and DNA extraction with QuickExtract™ FFPE DNA Extraction Kit (Lucigen®), (2) quantification with InnoQuant® HY (InnoGenomics Technologies) for sensitive assessment of total human and male DNA quantity/quality, and (3) massively parallel sequencing for simultaneous genotyping of 231 short tandem repeat (STR) and single-nucleotide polymorphism (SNP) markers with the ForenSeq® DNA Signature Prep Kit (Verogen, Inc.). Initial evaluation of artificially degraded blood samples (n = 10) achieved highly sensitive and informative quantification results with InnoQuant® HY, enabling successful first pass genotyping with the MiSeq FGx® System. Twentythree STR alleles (out of 85) and 70 identity informative SNP loci (out of 94) were recovered from two pg total long target DNA input (0.86 ng total short target input) and an InnoQuant degradation index (DI) of 460 (severely degraded). The three-part workflow was subsequently applied to teeth samples (dental pulp, root cement tissues; n = 13) with postmortem intervals (PMI) of the teeth ranging from 8 days to approximately 6 months. Informative SNP and STR DNA profiles were obtained, to include 78 STR alleles and 85 identity informative SNP loci typed (of 94 total SNP targets) in a 1 month, four-day PMI root cement sample with one pg total long target DNA input and a DI of 76. These data indicate successful performance of the proposed workflow from degraded DNA from teeth samples. P. Carrasco, C. Inostroza and M. Didier contributed equally to this work.

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“…TEs are not only a powerful tool for phylogeny and population genetics [33][34][35], but are also known to be associated with several diseases [21,36] and were used in a very recent forensic study dealing with quantity and sample quality in human DNA samples [37]. Large-scale analyses have already shown contributions to human genomic diversity [38][39][40].…”

Section: Introductionmentioning

confidence: 99%

Duplex Alu Screening for Degraded DNA of Skeletal Human Remains

Haß¹,

Hummel²,

Piskurek³

2017

Diversity

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Abstract:The human-specific Alu elements, belonging to the class of Short INterspersed Elements (SINEs), have been shown to be a powerful tool for population genetic studies. An earlier study in this department showed that it was possible to analyze Alu presence/absence in 3000-year-old skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. We developed duplex Alu screening PCRs with flanking primers for two Alu elements, each combined with a single internal Alu primer. By adding an internal primer, the approximately 400-500 bp presence signals of Alu elements can be detected within a range of less than 200 bp. Thus, our PCR approach is suited for highly fragmented ancient DNA samples, whereas NGS analyses frequently are unable to handle repetitive elements. With this analysis system, we examined remains of 12 individuals from the Lichtenstein cave with different degrees of DNA degradation. The duplex PCRs showed fully informative amplification results for all of the chosen Alu loci in eight of the 12 samples. Our analysis system showed that Alu presence/absence analysis is possible in samples with different degrees of DNA degradation and it reduces the amount of valuable skeletal material needed by a factor of four, as compared with a singleplex approach.

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Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets

Cited by 27 publications

References 27 publications

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing

Duplex Alu Screening for Degraded DNA of Skeletal Human Remains

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