The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

Fattorini, Paolo; Previderé, Carlo; Sorçaburu-Cigliero, Solange; Marrubini, Giorgio; Alù, Milena; Barbaro, A.; Carnevali, E.; Carracedo, Ángel; Casarino, L.; Consoloni, Lara; Corato, S.; Domenici, Ranieri; Fabbri, M.; Giardina, Emiliano; Grignani, Pierangela; Baldassarra, Stefania Lonero; Moratti, Marco; Nicolin, Vanessa; Pelotti, Susi; Piccinini, Andrea; Pitacco, Paola; Plizza, Laura; Resta, Nicoletta; Ricci, U.; Robino, Carlo; Salvaderi, Luca; Scarnicci, Francesca; Schneider, Peter M.; Seidita, Gregorio; Trizzino, Lucia; Turchi, Chiara; Turrina, Stefania; Vatta, Paolo; Vecchiotti, Carla; Verzeletti, Andrea; Stefano, Francesco De

doi:10.1002/elps.201400141

Cited by 12 publications

(27 citation statements)

References 36 publications

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“…To enrich the samples in A‐P sites, a method previously published was followed . Briefly, 200 μL aliquots, each one containing about 13 μg of DNA, were incubated in a water bath at 70°C for 6.0, 8.0 and 10.0 hours (namely sample FM‐6, FM‐8 and FM‐10, respectively).…”

Section: Methodsmentioning

confidence: 99%

Performance of the ForenSeq^TM DNA Signature Prep kit on highly degraded samples

et al. 2017

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Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeq DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub-optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9-10.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as "low coverage" (< 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeq DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1-44.8% and 10.9-58.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics.

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Section: Methodsmentioning

confidence: 99%

Performance of the ForenSeq^TM DNA Signature Prep kit on highly degraded samples

et al. 2017

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“…In fact, the ability of calling the STR loci decreased from 46 to 17% proportionally to the degradation of the trial samples and similarly from 68 to 26% for the SNP loci. In addition, correct typing according to the interpretation guidelines described in [5] was achieved from 39 to 4% of the STRs and from 55 to 13% of the SNPs when the genotypes from the trial samples were compared to the ones recovered from the unmodified control DNA (see Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…1. Evaluation of the genotypes according to [5]. Y axis: frequency of the loci correctly typed (C), of the loci which gave no results (NR), ambiguous (U) or wrong typing (W).…”

Section: Discussionmentioning

confidence: 99%

“…15 mg of DNA, as assessed by Nanodrop, were artificially degraded by aqueous hydrolysis at 37 C for 6 h, 8.5 h and 10 h using a protocol already described [5] with minor changes.…”

Section: Dna Depurination Protocolmentioning

confidence: 99%

“…DNA was extracted using a standard organic extraction protocol based on phenol/chloroform purification and ethanol precipitation [5].…”

Section: Dna Extractionmentioning

confidence: 99%

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Evaluation of the reliability of the data generated by Next Generation Sequencing from artificially degraded DNA samples

Carboni

Fattorini

Previderé

et al. 2015

Forensic Science International: Genetics Supplement Series

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NGS has the potential to be a promising technology for recovering genetic information from challenging\ud specimens in forensic genetics. In order to understand the role of DNA damage on the outcome of NGS, we\ud investigated the performance of ForenSeqTM DNA Signature kit, Illumina (in its pre-commercial version)\ud on a set of in vitro degraded trial DNA samples. After DNA quantification by qPCR, duplicate analyses of\ud the samples were carried out. The resulting molecular products were then sequenced by using MiSeq1\ud system (Illumina) and analyzed using ForenSeqTM Universal Analysis Software (Illumina). The coverage\ud and error rate of the NGS data obtained from the degraded samples were compared to the ones gathered\ud from the unmodified DNA. The NGS data showed that the ability of recovering genotypes and the\ud frequency of analytical artifacts are strongly influenced by the degree of damage of the template. NGS was\ud able to call 46–17% of the STR loci and 68–26% of the SNPs in the degraded samples. In addition, when the\ud genotypes from the degraded samples were compared to the ones recovered from the unmodified control\ud DNA, correct typing was achieved from 39 to 4% of the STRs and from 55 to 13% of the SNPs.\ud These data show that NGS is a powerful method for gathering genetic data from samples which failed\ud the conventional approaches, even if in this experiment the risk of mistyping seems not to be negligible\ud (up to 2%)

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Isometric artifacts from polymerase chain reaction‐massively parallel sequencing analysis of short tandem repeat loci: An emerging issue from a new technology?

et al. 2022

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The recent introduction of polymerase chain reaction (PCR)‐massively parallel sequencing (MPS) technologies in forensics has changed the approach to allelic short tandem repeat (STR) typing because sequencing cloned PCR fragments enables alleles with identical molecular weights to be distinguished based on their nucleotide sequences. Therefore, because PCR fidelity mainly depends on template integrity, new technical issues could arise in the interpretation of the results obtained from the degraded samples. In this work, a set of DNA samples degraded in vitro was used to investigate whether PCR‐MPS could generate “isometric drop‐ins” (IDIs; i.e., molecular products having the same length as the original allele but with a different nucleotide sequence within the repeated units). The Precision ID GlobalFiler NGS STR panel kit was used to analyze 0.5 and 1 ng of mock samples in duplicate tests (for a total of 16 PCR‐MPS analyses). As expected, several well‐known PCR artifacts (such as allelic dropout, stutters above the threshold) were scored; 95 IDIs with an average occurrence of 5.9 IDIs per test (min: 1, max: 11) were scored as well. In total, IDIs represented one of the most frequent artifacts. The coverage of these IDIs reached up to 981 reads (median: 239 reads), and the ratios with the coverage of the original allele ranged from 0.069 to 7.285 (median: 0.221). In addition, approximately 5.2% of the IDIs showed coverage higher than that of the original allele. Molecular analysis of these artifacts showed that they were generated in 96.8% of cases through a single nucleotide change event, with the C > T transition being the most frequent (85.7%). Thus, in a forensic evaluation of evidence, IDIs may represent an actual issue, particularly when DNA mixtures need to be interpreted because they could mislead the operator regarding the number of contributors. Overall, the molecular features of the IDIs described in this work, as well as the performance of duplicate tests, may be useful tools for managing this new class of artifacts otherwise not detected by capillary electrophoresis technology.

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The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

Cited by 12 publications

References 36 publications

Performance of the ForenSeq^TM DNA Signature Prep kit on highly degraded samples

Performance of the ForenSeq^TM DNA Signature Prep kit on highly degraded samples

Evaluation of the reliability of the data generated by Next Generation Sequencing from artificially degraded DNA samples

Isometric artifacts from polymerase chain reaction‐massively parallel sequencing analysis of short tandem repeat loci: An emerging issue from a new technology?

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The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

Cited by 12 publications

References 36 publications

Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples

Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples

Evaluation of the reliability of the data generated by Next Generation Sequencing from artificially degraded DNA samples

Isometric artifacts from polymerase chain reaction‐massively parallel sequencing analysis of short tandem repeat loci: An emerging issue from a new technology?

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Performance of the ForenSeq^TM DNA Signature Prep kit on highly degraded samples

Performance of the ForenSeq^TM DNA Signature Prep kit on highly degraded samples