Validation study of STR analysis from buccal swab by 3500xL Genetic Analyzer using an AmpFlSTR^® Identifiler^® PCR Amplification Kit

Abstract

  The 3500xL Genetic Analyzer will be used for short tandem repeat (STR) analysis because 3130xl Genetic Analyzer has been discontinued. A validation study was performed for the 3500xL to carry out STR analysis of reference samples with Identifiler^® Kit.
  Sensitivity test was performed with 0.125 ng, 0.5 ng, 1 ng and 3 ng DNA as PCR template. All alleles were detected with 0.5 ng, 1 ng and 3 ng DNA input. Any off scale data and noise over 175 RFU that is threshold value of 3500xL recommended by the manufacturer were not observed from 0.5 ng of input DNA. Thus the optimum input DNA for PCR template was 0.5 ng. We also examined Intra-color peak height ratio and heterozygote peak height ratio at 0.5 ng of template DNA. These results obtained by 3500xL showed similar validations with 310 and 3130xl previously reported. Spectral pull-up was examined by inspections of 240 injections data about each 0.5 ng, 1 ng and 3 ng DNA samples in the sensitivity study with a threshold value of 50 RFU. 1,011, 3,210, and 7,056 pull-up peaks were observed in 0.5 ng, 1 ng, and 3 ng, respectively. It was possible to remove all pull-up peaks using a global cut-off filter of 20%. We compared the profiles generated by the 3500xL and 3130xl using 84 samples with 0.5 ng DNA as PCR template. Both 3500xL and 3130xl detected peaks of all alleles contained in them. However, 3500xL also detected noise peaks and genotyping success rate were 71.4-88.1%. When the 3500xL run data were re-analyzed using the 20% filter, the genotyping results showed 100% concordance.
  The 20% filter is useful to obtain STR profiles with Identifiler^® kit using 3500xL from reference samples because reference samples yield high-quality DNA and are collected from a single individual.