A New Multiplex Assay of 17 Autosomal STRs and Amelogenin for Forensic Application

This paper describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) systems for 17 autosomal loci (D1S1656, D2S441, D3S1358, D3S3045, D6S477, D7S3048, D8S1132, D10S1435, D10S1248, D11S2368, D13S325, D14S608, D15S659, D17S1290, D18S535, D19S253 and D22-GATA198B05) and Amelogenin. Primers for the loci were designed and optimized so that all of the amplicons were distributed from 50 base pairs (bp) to less than 500 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. Strategies were developed to overcome challenges that encountered in creating the final assay. The limits of the multiplex were tested, resulting in the successful amplification of genomic DNA range from 0.25–4 ng with 30 PCR cycles. A total of 681 individuals from the Chinese Han population were studied and forensic genetic data were present. No significant deviations from Hardy–Weinberg equilibrium were observed. A total of 180 alleles were detected for the 17 autosomal STRs. The cumulative mean exclusion chance in duos (CMECD) was 0.999967, and cumulative mean exclusion chance in trios (CMECT) was 0.99999995. We conclude that the present 17plex autosomal STRs assay provides an additional powerful tool for forensic applications.


Introduction
The study of genetic variation, using DNA polymorphisms distributed throughout the genome, has allowed better understanding of the history and diversity of human populations as well as providing a system for forensic testing [1,2]. At present, STR typing can be considered a standard approach and the method of choice in the forensic field, allowing a high discrimination power adequate for addressing most problems of human identification and paternity testing [3]. Analysis of STR loci is an essential component of forensic genetics. 13 core STR loci of Combined DNA Index System (CODIS) which used for a DNA database of felon were widely used in commercial kits [4]. On the other hand, many highly polymorphic STR loci exist, which are unlinked to the core STR loci (non-CODIS loci). Analyses of such polymorphic non-CODIS loci in addition to the core CODIS loci may increase the power of exclusion in complex kinship testing [4,5].

Ethics Statement
Human blood samples were collected with the approval of the Ethics Committee of the Institute of Forensic Sciences, Ministry of Justice, P.R. China. Samples were obtained from unrelated volunteers after receiving written informed consents. Animal blood samples were collected with the approval of the Animal Use Committee of the Institute of Forensic Sciences, Ministry of Justice, P. R. China.

(a) Candidate STRs Selection
The initial candidate pool of autosomal STRs for this study was based on the STR DNA Internet DataBase created by John M. Butler et al (http://www.cstl.nist.gov/strbase/) and National Center for Biotechnology Information (NCBI) (http://www.ncbi. nlm.nih.gov/). STRs were then selected according to the following criteria: (I) non-coding, non-CODIS, autosomal STRs; (II) average heterozygosity $0.30 in European, African and Asian population groups; (III) located on different chromosomes, if not, at most two STRs were selected from the same chromosome and make sure on different arms; and (IV) physical distance between the selected STRs and CODIS STRs above 10 Mb.
After applying the described criteria, 16 ideal candidate STRs were obtained. Flanking sequences of these STRs, as well as reported sequence variants within these region, were obtained using the University of California Santa Cruz Genome Browser (Human February 2009 Assembly; GRCh37/hg19) at http:// genome.ucsc.edu/.
Locus D3S1358 which belonged to CODIS STRs was added to our final assay, for comparing the accuracy of the final multiplex and usually used commercial kit (e.g. AmpF,STRH IdentifilerH Kit). The sex-typing locus Amelogenin was also included in the final assay.

(b) Primer and Multiplex Assay Design
PCR primer designing was performed using software of primer premier v5.0 and Oligo v6.0, applying the following main criteria: primer length of 18-30 bp; amplicon length of 50-500 bp; optimum Tm from 56uC to 64uC; optimum CG content range from 45% to 55%. Obtained primer pairs were then checked for non-specific hybridizations in other genome regions using NCBI Basic Local Alignment Search Tool (BLAST) at http://blast.ncbi. nlm.nih.gov/. AutoDimer software was also applied to screen candidate primer sequences for inter-primers compatibility.
Forward primer of each STR was labelled at the 59 end with either 6 FAM TM , HEX TM , TEMRA TM and ROX TM (Applied Biosystems, Foster City, CA). All primers were synthesized and labelled by Sangon Biotech. Co. Ltd (Shanghai, China). All selected STRs were then organized by expected amplicon length and assigned into four different dye-labelling fluorochromes in order to achieve an evenly balanced genotyping assay for a single PCR and electrophoretic separation.

(c) PCR Amplification and STRs Detection
All selected STRs were initially amplified in singleplex, in order to evaluate primer performance and expected allele sizes. After optimization, the amplification of all STRs was performed in a single multiplex PCR including 16PCR buffer, 200 mM dNTP, 1.5 mM MgCl 2 , 1 U GoldTaq DNA polymerase, 5 mL primer mix and 0.25-4 ng of genomic DNA in a 25 mL final reaction volume. Thermocycling condition for the multiplex PCR assay was initial incubation at 95uC for 11 min; 30 cycles at 94uC for 30 s, 60uC for 60 s and 70uC for 60 s; with a final extension at 60uC for 30 min.
PCR products were subsequently prepared for capillary electrophoresis by adding 1 mL of each amplified product to 9 mL of a 17:1 mixture of Hi-Di formamide and Orange-500 size standard, respectively. Separation and detection were performed with AB 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA) using filter set E5 and POP4 polymer (Applied Biosystems, Foster City, CA). Samples were injected electrokinetically for 5 sec at 3 kV. The STR alleles were then separated at 15 kV at a run temperature of 60uC. Genotyping data of studied samples were then collected with GeneMapper v3.2.1 software (Applied Biosystems, Foster City, CA). Positive control DNA of 9947A human cell line samples (Applied Biosystems, Foster City, CA) was used to test the overall performance of the multiplex genotyping assay.

(d) Sensitivity Testing
To determine the minimum quantity of DNA required to achieve reliable results (peak highs at all loci exceeded 100 relative  fluorescence units (RFU)) with the multiplex assay, we used aliquots of the cell line 9947A DNA (50, 100, 250, 500 pg, 1, 2, 4 and 5 ng). Every concentration tested for three replicates.

(e) Specificity Testing
To determine the specificity of this multiplex assay, several kinds of living creatures' bloods (Human, Pig, Chick, Duck, Dog and Cat) were tested.

(f) Population Samples for STR Polymorphism Analysis
Peripheral bloods were collected from a total of 681 unrelated healthy individuals (343 males and 338 females) of Chinese Han population, the largest nation of China. All samples of the volunteers were genotyped with commercial kit of AmpF,STRH IdentifilerH PCR Amplification Kit before.
Genomic DNA was extracted using the Chelex-100 protocol as described by Walsh et al [6]. The quantity of recovered DNA was determined by spectrophotometric method.

(a) Primer and Multiplex PCR Optimization
The primer sequences, T m values and concentrations of the 17 autosomal STR loci and Amelogenin locus in the final multiplex assay were listed in Table 1. Primers for each STR locus were initially tested in singleplex to evaluate the performance. The criteria for primer ''failure'' in this study is defined as those that produce profiles that exhibit incomplete adenylation, the presence of PCR artifacts, nonspecific products, low signal, or no PCR product at all [5]. With those ''failure'' ones, singleplex PCR optimization were adopted, otherwise, re-designing the PCR primers. Once the successful primers at locus were determined, those were combined together and tested in multiplex. At first, the tested multiplex assay indicated a high degree of baseline noises and many profiles displayed the features of failure. This was due to multiplex primer incompatibility. Based on the data of primer concentrations and peak heights, optimizations of the multiplex assay were performed.
The final assay design resulted in a good working base to set up the multiplex reaction. In the initial multiplex tests some primer pairs revealed weaker performance than others, as would be expected since the optimum annealing temperature was not exactly the same for all primers. To minimize these effects and aiming for a more balanced result, we tested different primer mix concentrations and annealing temperatures. After the multiplex development and optimization, 17 autosomal STRs plus Amelogenin were successfully amplified in a single PCR reaction, following the final optimum conditions. The genotyping profiles of positive control DNA of 9947A and ladder were showed in Figure 1 and Figure 2, respectively.

(b) Multiplex PCR Performance
In general, during the genotyping of 681 unrelated population samples, the multiplex reaction revealed to be robust and able to successfully amplify all STRs in samples with 0.25-4 ng DNA in a 25 mL final reaction volume. The genotyping results of locus D3S1358 by our new multiplex assay and the commercial kit of AmpF,STRH IdentifilerH Kit were identical with each other.
Several DNA concentrations ranging from 50 pg to 5 ng were tested with the multiplex, using dilution series of positive control sample 9947A. The best results, revealing an improved peak balance among STRs, were observed when using 0.5-1 ng of DNA in a 25 mL PCR final volume, although good results for all STRs were obtained for concentrations from 0.25 to 4 ng. Nevertheless, when using highly concentrated DNA samples (.4 ng), we observed strong signals and over-scaled peaks; in these cases, the presence of pull-ups and abnormally shaped peaks due to ineffective fluorescence correction can make interpretation of the genotyping results difficult. Furthermore, the use of higher amounts of DNA in the reaction occasionally caused the inhibition of amplification of longer size amplicons. In samples with DNA amounts as low as 0.1-0.25 ng, it was still possible to amplify all markers; nonetheless, the results were not always consistent, sometimes presenting unusual allele imbalances or allele dropouts observed from the known consensus genotypes of the control DNAs used. The marked heterozygote imbalance or allele dropouts with small quantities of target DNA have been previously reported and are a consequence of stochastic variation in PCR amplification when the number of template molecules is very low [9]. We highlight the fact that, in low copy number applications of this multiplex, the results must be interpreted with caution, and duplicate analyses are required to minimize errors.
As a practical application for forensic use, the multiplex assay was employed in several kinds of living creatures' bloods (Human, Pig, Chick, Duck, Dog, Cat), aiming to confirm the specificity. Only human blood can have positive results. There are no detected signals for other creatures at any of these 18 STRs included in the kit, which proved that this kit is only suitable for human testing.
Genetic linkage disequilibrium (LD) in the human genome would not for consideration if the physical distances are more than 10 Mb [10,11,12]. Thus, LD between the current 16 non-CODIS STR and the CODIS core loci may be excluded from consideration. Analysis of extended STR loci using both the present multiplex system and commercial kits which include the CODIS core loci have been demonstrated to be remarkably effective in cases involving difficult kinship testing.

(d) Forensic Efficiency in Han Populations
The polymorphism analysis of 17 autosomal STRs was studied in the Han population of China. All studied STRs followed Hardy-Weinberg equilibrium. In Table 2 we present allele frequencies derived from the population and the forensic efficiency Table 2. Frequencies and forensic parameters of 17 autosomal STRs.  Since all of the 17 autosomal STR loci were independent from each other, the combined forensic efficiency parameters were calculated based on allele frequencies while cumulative mean exclusion chance in duos (CMEC D ) was 0.999967, and cumulative mean exclusion chance in trios (CMEC T ) was 0.99999995. These results suggest that the 17 autosomal STR loci included in the multiplex assay are highly polymorphic and informative in the Han population of China.

Concluding Remarks
In this study, we displayed a simple and sensitive 17plex STRs new assay for forensic application, which allows the simultaneous genotyping of 17 autosomal STRs plus Amelogenin presenting high polymorphic in Chinese Han population. The development of this 17plex assay provides support for the use of additional loci for complex kinship testing involving deficient paternity cases and for use in missing person/mass disaster cases.