| The China forensic science DNA database has become the largest database in the world in a relatively short time. Up to Dec 30, 2014, there are more than 34 million profiles in DNA database. The main reagent for establishing forensic science DNA database is STR multiplex kits. The initial STR kits used in China are imported and manufactured by Applied Biosystem or Promega. However, several domestic commercial multiplex kits have been developed, such as DNA Typer®15 Plus, AGCU17+1, and Goldeneye TM 20 A. Because different kits may contain use different sets of PCR primers for the same loci, or the primer binding region was mutated, the discordant genotypes have been observed.To investigate the discordant rate of using eight general STR multiplex kits for STR typing of samples contained within the China forensic science DNA database, 2203 unrelated individuals of the Han population in China were analyzed with 5 original import multiplex kits(i.e., Identifiler TM, Identifiler® Direct, Identifiler® Plus, Sinofiler TM,Power Plex®16HS) and 3 domestic commercial multiplex kits(i.e., DNA Typer®15 Plus,AGCU 17+1, Goldeneye TM 20A), total 20 STR loci, to observe typing results.Statistically obtained 19 STR loci were investigated for population genetics polymorphism, to provide population genetics parameters for the forensic personal identification and paternity testing based on China forensic science DNA databases; on which samples with disconcordant typing results, leading to different STR typing or amplification with serious imbalance, were analyzed by sequencing, to find out the mutation point, analyze causes of the disconcordance. The concordance test was employed to assess effects of different typing results of the same sample by different STR kits on the construction of China forensic science DNA databases, to provide ideas for the standardization of the construction of China forensic science DNA database.Results:1. Distributions of 19 STR loci of 2203 unrelated Han ethnic Chinese individuals are all in accord with Hardy-Weinberg balance(P >0.05), having high polymorphism, in which the polymorphism of Penta E locus in the highest. For 19 gentic markers, the heterozygosity(H) ranged from 0.570 to 0.921, the probability of discrimination power(DP) ranged from 0.770~0.987, the polymorphism information content(PIC) ranged from 0.52 to 0.91, and the excluding probability of paternity(PE) ranged from 0.257 ~0.838. The total probability of discrimination power(TDP) was 1-9.57×10-24 and the cumulative probability of exclusion(CPE)reached 1-1.20×10-8.2. Amplified results of 2203 samples by 8 different kits were compared; typing differences were found in every kind of kit and 35 cases. Disconcordance rate was1.589% for results.3. For comparison of kits in pairs, AGCU 17+1 kit and Goldeneye TM 20 A kit have the highest disconcordance of detecting results, which was 0.999%. The concordance of comparison results between Amp Fl STR series kits, and between Powerplex® 16 HS kit and Goldeneye TM 20 A kit, are all 100%.4. There were differences of typing results in 35 disconcordance samples, in which was allele dropout in 23 samples; was difference of typing results in 5 samples; was serious imbalance of amplification in 7 samples; were allele dropout and serious imbalance of amplification simultaneously in 1 sample. Sequencing results showed that there were 4base lost at D19S433 locus in 5 samples with difference of typing results, no mutations in 3 samples with serious imbalance of amplification, possible undetected mutations in5 samples with allele dropout, mutation points in 22 samples.5. The disconcodance rate of detection results was 1.589% of 8 different kits, with confidence interval 1.11% to 2.2%. Calculation based on the amount of 34 million items on China forensic science DNA database, there are up to 748,000 samples with disconcordance.6. The expected weighted average difference rate is 0.5% for comparison in pairs. The false exclusion rate is 0.104% and may be 8,120 records that should have been matched but were not matched because of difference between kits in China forensic science DNA database in 2014.Conclusion:1. 19 STR loci of 8 commonly used kits for the construction of China forensic science DNA databases have the nice individual recognizing ability for Chinese Han population,suitable for forensic individual identification and paternity test of Chinese Han population.2. 8 kinds of kits for 5 companies were used for the construction of China forensic science DNA databases. Disconcordant results were mainly caused by different primers used by different kits of different companies. AB company use the same primer to ensure the concordance of results. Goldeneye TM 20 A kits may use the same primer as Powerplex® 16 HS kits. The existence of nucleotide polymorphism mutation on the proximity to 3’ end of primer binding site is the major reason of allelic loss. Bases loss of the lateral wing sequence on the central repeat region is the cause of different typing results. Sequence mutations on the 3’ end of primer binding site or low amount of template DNA can lead to serious imbalance of amplification.3. The false exclusion rate is 0.104% and the effects of disconcirdance of detection results by different STR multiplex amplification kits on DNA database should arouse enough attention. When practically establishing the database, a unified STR amplification kits should be tried to use to avoid disconcordance. A loose standard should be applied when comparing the same type, trying to avoid typing mistakes caused by allelic dropout or amplification inbalance. |
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