Assessment of the Precision ID Identity Panel kit on challenging forensic samples

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the \u201csame donor\u201d or from a \u201cfirst degree relative\u201d was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21-26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine\u2122 (PGM\u2122) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50% and 35% of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18-23% when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37% of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 x 10-13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping

Highly degraded RNA can still provide molecular information: An in vitro approach

10siThe long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical–chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat-mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di-ethyl-pyro-carbonate-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r2 > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end-point PCR of blood-specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR-based results show that RNA maintains the ability to be retro-transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long-term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed.partially_openopenFattorini P.; Bonin S.; Marrubini G.; Bertoglio B.; Grignani P.; Recchia E.; Pitacco P.; Zupanic Pajnic I.; Sorcaburu-Ciglieri S.; Previdere C.Fattorini, P.; Bonin, S.; Marrubini, G.; Bertoglio, B.; Grignani, P.; Recchia, E.; Pitacco, P.; Zupanic Pajnic, I.; Sorcaburu-Ciglieri, S.; Previdere, C

Highly degraded RNA can still provide molecular information: An in vitro approach

The long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical–chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat-mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di-ethyl-pyro-carbonate-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r2 > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end-point PCR of blood-specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR-based results show that RNA maintains the ability to be retro-transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long-term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed

Systematic study on the analytical parameters relevant to achieve reliable STR profiles, as assessed in a multicentre data set.

A set of 16 amplifications following a D-optimal experimental design was planned using the most commonly selected kit and sequencer utilized in a collaborative exercise of the ISFG Italian Working Group GeFI on the molecular characterization of a depurinated DNA sample. The data were evaluated using STRvalidator in order to describe the occurrence of PCR artifacts such as peak imbalance and allele/locus drop outs. The aim of the study was to describe the role of the template amount, number of amplification cycles, volume of the PCR reaction in determining the assay sensitivity and the profiling accuracy as measured by peak heights and areas. The results showed that only by selecting an appropriate ratio of template to PCR reaction solution total volume together with an increase in the number of cycles it could be possible to obtain balanced peaks as well as minimal occurrence of other artifacts. These data are useful to model and understand the PCR artifacts occurrence described in the collaborative exercise

Assessment of the Precision ID Identity Panel kit on challenging forensic samples

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the “same donor” or from a “first degree relative” was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21–26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine™ (PGM™) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18–23 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 × 10−13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping

Assessment of the Precision ID Identity Panel kit on challenging forensic samples

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the \u201csame donor\u201d or from a \u201cfirst degree relative\u201d was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21\u201326 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine\u2122 (PGM\u2122) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18\u201323 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 7 10 1213, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping

Assessment of the Precision ID Identity Panel kit on challenging forensic samples

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the “same donor” or from a “first degree relative” was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21–26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine™ (PGM™) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18–23 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 × 10−13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping