title: Revealing the challenges of low template DNA analysis with the prototype Ion AmpliSeq™ Identity panel v2.3 on the PGM™ Sequencer.
abstract: Forensic scientists frequently have to deal with the analysis of challenging sources of DNA such as degraded and low template DNA (LtDNA). The capacity to genotype difficult biological traces has been facilitated by emerging technologies. Massive parallel sequencing (MPS) on microchip among other technologies promises high sensitivity and discrimination power. In this study we evaluated the combined use of the Quantifiler(®) Trio DNA Quantification Kit with the prototype Ion AmpliSeq™ Identity panel v2.3 and PGM™ platform in LtDNA samples. Coverage, allele balance, allele drop-out/in, consistency and variance were assessed. Overall, the results showed a great level of performance and consistency in terms of genotyping capability even under the most challenging conditions, making it possible to obtain consistent SNP profiles with 31 pg of DNA and partial informative profiles with as little as 5 pg or with severely degraded DNA. In addition, we demonstrated that the stochastic effects observed in some samples are due to the amplification of the library rather than sequencing. Based on our data, we proposed general recommendations for the analysis of casework samples starting from the use of quantification data, which proved to be critical in deciding whether to process the samples via STR (short tandem repeat) analysis or SNP MPS. In our experience, the use of the prototype Ion AmpliSeq™ Identity panel v2.3 has revealed a new applicable solution for processing LtDNAs. This approach provides users with an additional tool for analysis of traces that either would not give informative results with conventional STR-based techniques.
authors.affiliation: Carabinieri, Investigazioni Scientifiche di Roma, Sezione di Biologia, Rome, Italy. Electronic address: elena.salata@carabinieri.it.
authors.forename: Salata
authors.initials: S
authors.lastname: Elena
authors.affiliation: Thermo Fisher Scientific, Monza, Italy. Electronic address: slessandro.agostino@thermofisher.com.
authors.forename: Agostino
authors.initials: A
authors.lastname: Alessandro
authors.affiliation: Carabinieri, Investigazioni Scientifiche di Roma, Sezione di Biologia, Rome, Italy.
authors.forename: Ciuna
authors.initials: C
authors.lastname: Ignazio
authors.affiliation: Thermo Fisher Scientific, South San Francisco, CA, USA. Electronic address: sharon.wootton@thermofisher.com.
authors.forename: Wootton
authors.initials: W
authors.lastname: Sharon
authors.affiliation: Carabinieri, Investigazioni Scientifiche di Roma, Sezione di Biologia, Rome, Italy.
authors.forename: Ripani
authors.initials: R
authors.lastname: Luigi
authors.affiliation: Carabinieri, Investigazioni Scientifiche di Roma, Sezione di Biologia, Rome, Italy.
authors.forename: Berti
authors.initials: B
authors.lastname: Andrea
mesh_terms: D000483:Alleles; D004247:DNA / Q000032:analysis* / Q000097:blood / Q000235:genetics; D053803:Forensic Genetics / Q000379:methods*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA
keywords: Ion AmpliSeq™ Identity panel; LtDNA; Massive parallel sequencing; PGM™ Sequencer; Quantifiler(®); STR analysis