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

Carboni, Ilaria; Fattorini, Paolo; Previderé, Carlo; Ciglieri, Solange Sorçaburu; Iozzi, S.; Nutini, A.L.; Contini, Elisa; Pescucci, Chiara; Torricelli, Francesca; Ricci, U.

doi:10.1016/j.fsigss.2015.09.034

Cited by 5 publications

(8 citation statements)

References 5 publications

(8 reference statements)

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“…As reported in Tables and , the analysis of STR and SNP data showed a high percentage of loci which gave none or less than 50 reads in the three trial samples (up to 75.7–76.1% in sample FM‐10) and a positive trend between the extent of DNA degradation and the % of low‐covered loci. Although this setting of the cut‐off value is arbitrary, and leads to a loss of genetic information of up to 5.7% (when compared to the cut‐off of 30 x) , we believe that a conservative approach focused on the reliability of the results is what is needed in forensic genetics when validation studies are underway to better understand the potential of an innovative technique.…”

Section: Resultsmentioning

confidence: 99%

“…The only pivot study performed with the ForenSeq TM DNA Sequencing kit reported a minimum DoC of 10x . Our preliminary data analysis, performed with a cut off of 30x, evidenced mistyping error rates of up to 2.2% in these three trial samples . For this reason, we tried to reduce these error rates by increasing the minimum coverage for “locus call” to 50x.…”

Section: Methodsmentioning

confidence: 92%

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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: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 92%

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

et al. 2017

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“…Reliable and reproducible results were produced from high-concentration single-source samples and profiles could be differentiated from mixed samples (Churchill et al, 2016;Iozzi et al, 2015). Low concentration, degraded and case type samples produced results concordant with CE data (Carboni et al, 2015;Churchill et al, 2016). The BGA and EVC SNPs were also tested and found to accurately predict the ancestry, hair and eye color of European and East Asian samples (Hussing, Borsting, Mogensen, & Morling, 2015).…”

Section: The Miseq Fgx™ Forensic Genomics Solutionmentioning

confidence: 90%

“…After a number of studies completed assessments of the MFGS (Caratti, Turrina, Ferrian, Cosentino, & De Leo, 2015;Carboni et al, 2015;Churchill et al, 2016;Hussing et al, 2015;Iozzi et al, 2015;Just, Moreno, Smerick, & Irwin, 2017), Jäger et al (2017) published the developmental validation of the MFGS. Since then others have completed internal and inter laboratory studies (Köcher et al, 2018;Laurent et al, 2017;Moreno et al, 2018).…”

Section: Validationmentioning

confidence: 99%

A review of the method and validation of the MiSeq FGx™ Forensic Genomics Solution

England

Harbison

2019

WIREs Forensic Science

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The MiSeq FGx™ Forensic Genomics Solution (MFGS; Verogen Inc.) is a massively parallel sequencing workflow using Illumina sequencing technology. The workflow includes the ForenSeq™ DNA Signature Prep kit, the MiSeq FGx™, and the ForenSeq™ Universal Analysis Software (UAS). This review explores the molecular biology and bioinformatic methods used during the preparation of samples into sequencing libraries, the MiSeq FGx™ sequencing process, and the data analysis. We highlight what happens during each of these steps and how they influence the final results, while identifying limitations and possible improvements. After initial evaluations established its suitability for forensic applications, a number of studies have completed forensic validation studies. We review this work, and find the MFGS has shown to be a reliable and robust technology. The successful validation of the solution has allowed its implementation into forensic casework in a number of jurisdictions. Some questions still remain around the normalization of the sequencing libraries, and the bioinformatic processing and analysis of the sequencing results. In particular, the markers for biogeographical ancestry and externally visible characteristics require further research into their performance, and the ForenSeq™ UAS, in its current form, is limited in its ability to accurately predict these characteristics. We suggest a number of alternative bioinformatic tools that could be used instead.

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“…Moreover, since the DNA fragments do not have to be separated by size, the amplicon length can be reduced, which benefits the analysis of degraded samples [1,19]. Additionally, the sequencing of SNPs can provide valuable information if CE-based STR typing fails [4].…”

Section: Introductionmentioning

confidence: 99%

Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples from altered human corpses using the MiSeq FGx system

Senst¹,

Caliebe²,

Scheurer³

et al. 2022

Journal of Forensic Sciences

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The proceeding developments in next generation sequencing (NGS) technologies enable increasing discrimination power for short tandem repeat (STR) analyses and provide new possibilities for human identification. Therefore, the growing relevance and demand in forensic casework display the need for reliable validation studies and experiences with challenging DNA samples. The presented validation of the MiSeq FGx system and the ForenSeq™ DNA Signature Prep Kit (1) investigated sensitivity, repeatability, reproducibility, concordance, pooling variations, DNA extraction method variances, DNA mixtures, degraded, and casework samples and (2) optimized the sequencing workflow for challenging samples from human corpses by testing additional PCR purification, pooling adjustments, and adapter volume reductions. Overall results indicate the system's reliability in concordance to traditional capillary electrophoresis (CE)‐based genotyping and reproducibility of sequencing data. Genotyping success rates of 100% were obtained down to 62.5 pg DNA input concentrations. Autosomal STR (aSTR) profiles of artificially degraded samples revealed significantly lower numbers of locus and allelic dropouts than CE. However, it was observed that the system still exposed drawbacks when sequencing highly degraded and inhibited samples from human remains. Due to the lack of studies evaluating the sequencing success of samples from decomposed or skeletonised corpses, the presented optimisation studies provide valuable recommendations such as an additional PCR purification, an increase in library pooling volumes, and a reduction of adapter volumes for samples with concentrations ≥31.2 pg. Thus, this research highlights the importance of all‐encompassing validation studies for implementing novel technologies in forensic casework and presents recommendations for challenging samples.

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

Cited by 5 publications

References 5 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

A review of the method and validation of the MiSeq FGx™ Forensic Genomics Solution

Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples from altered human corpses using the MiSeq FGx system

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

Cited by 5 publications

References 5 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

A review of the method and validation of the MiSeq FGx™ Forensic Genomics Solution

Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples from altered human corpses using the MiSeq FGx system

Contact Info

Product

Resources

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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