Introduction

In early 1990s, the advent of the polymerase chain reaction (PCR) technology and the use of short tandem repeat (STR) polymorphism1 was a major breakthrough in forensic DNA technology. In last two decades, PCR-based STR typing has become a routine technique in the forensic investigations due to associated features such as specificity, sensitivity, feasibility to simultaneously amplify several loci (multiplexing) and automation2,3. Despite several improvements in the methodology, time required for the conventional forensic STR analysis could not be reduced and the process is still lengthy needing hours to several days for processing of most of the samples4. Conventional forensic DNA typing is a multistep process involving several steps of DNA extraction, quantification, amplification, genotyping and analysis (Fig. 1). These steps are not just time consuming but also result in loss of the valuable DNA5,6, which is already in minute quantities in the forensic samples. Also multiple processing steps need more human intervention which further increases the possibility of contamination and error. If these steps are reduced and the amplification could be achieved without the extraction, quantification, and concentration processes, higher yield of DNA could be achieved. Also, the chances of manual error and contamination could be reduced. Approximately 20–76% of DNA is lost from the swab samples during the DNA extraction step7. Various studies have reported that column-based DNA extraction techniques also result in the loss of DNA, thereby affecting the genotyping using this method8,9,10,11,12,13,14. Most of the time in routine forensic DNA typing is consumed in the pre-processing steps of DNA extraction and Quantitation4. The development of semi-automated DNA extraction kits and automated DNA extraction systems using pre-formulated kits has been a major breakthrough in this area so far. Some short non-automated extraction protocols like Chelex or alkaline lysis method and/or magnetic, paramagnetic beads based rapid DNA isolation are also in use. Since last decade, most of the forensic laboratories have encountered a considerable rise in the number of forensic DNA casework15,16,17. Towards the effort to avoid the delay in justice, a lot of research work has been conducted to improve the analysis speed and to develop faster processing methods to save the reporting time. Development of rapid, compact and portable devices capable of producing real-time results from forensic samples at the crime scene has been emphasised upon15,16,17. However, these efforts were not of much help to reduce the cost and time efficiency of the forensic DNA analysis, therefore were not adopted in most of the laboratories18. The analysis of mandatory reference samples in the forensic cases, which may vary from one to many, also require a similar lengthy processing time, as that of the case samples. This drawback is associated with both the methods viz. automated or specific direct amplification compatible chemistries19 which adds to the further delay in reporting the case. To curb down the analysis time and fasten the process of case reporting, there has been a growing interest towards direct amplification of case and reference samples such as blood and buccal swabs, single hair follicle and tape lifts of clothing, swab on FTA card20,21,22,23,24, blood25, fabrics6, hairs26,27, touch DNA28,29, blood stain30, fingernails31, tissues32,33 and fibers34. Direct amplification protocol eliminates the DNA extraction and quantitation steps, and accommodates the sample directly to the PCR step. The streamlined process of direct amplification for processing the DNA samples has obvious benefits such as simplify the process, low risk of contamination due to less handling steps, cost efficiency and has low chances of loss of forensic DNA, which already is in minute quantities32,33. Methods to successfully genotype different samples from varied origins in a single shot by direct amplification sounds promising and challenging too. Forensic cases have various sorts of circumstantial exhibits which could include anything found at the scene of crime. One such forensic case exhibits are buccal swabs and in the case, the subjects had a habit of smoking, chewing tobacco etc., it could cause inhibition in the process of PCR. Although, the buccal cells are stable for weeks or months if stored in the laboratory at – 20 °C till final processing, they are quite vulnerable to the bacterial growth, nucleic acid degradation. Reduced amplification ability after a span of 4 days or so has been observed, particularly under warm and moist conditions35. Keeping these points into consideration, saliva sample was chosen for the direct analysis. The fact that saliva or any cell suspension is expected to have some cell free DNA formed the ground for this study. Also, another important aspect considered was the nature and texture of the saliva cells as these cells are not strong enough to stand high temperatures like 95 °C, which is a prerequisite in most of the amplification kits for activation of hot start Taq polymerase36.

Figure 1
figure 1

Workflow of conventional forensic DNA typing.

Full size image

The process of genotyping nowadays does not require the high yield of DNA. This could be attributed to the improvement in the amplification step by using the upgraded and highly sensitive STR kits which require very low inputs of DNA (approx less than 500pg). The advantages associated with these advanced kits not only include the increase in the number of loci to be examined, as per the recommendation of the Scientific Working Group on DNA Analysis Methods (SWGDAM)37, the master mix has also been improved to reduce the amplification time and to cope with the PCR inhibitors. Rapid amplification of multiplex STRs for human identification has also been demonstrated by changing the master mix and/or enzyme22,23,38,39. There are certain multiplex kits specially designed for direct amplification of single source samples for the population data purpose. However, still most of the commonly used multiplex STR amplification kits need the pre-processing of samples before DNA amplification and genotyping40. Also, these specially designed kits vary in their markers. Therefore, if the standard kits are used for direct and rapid amplification, resultant profiles will not be comparable for all the tested markers with the profiles from routine casework samples and also from the existing DNA databases. Most of the widely used commercially available kits (non-direct) are not meant or reported to amplify the samples without DNA extraction. Working on a research project on X-STR’s, we collected saliva samples of unrelated individuals and used Investigator Argus X-12 multiplex STR kit (Qiagen, Germany) for DNA amplification as per manufacturer’s recommendation. DNA was isolated and extracted from 188 saliva samples using automated DNA extraction system 12 GC (Precision System Science Co., Ltd., Matsudo, Japan) following manufacturer’s instructions and pre formulated kits41. Few samples were tried for direct amplification of saliva samples with the same multiplex kit, using the same protocol for amplification. Promising results in terms of good quality DNA profile from the pilot study resulted into this detailed study on the direct amplification of saliva samples using commercially available non-direct multiplex kits. Kits used in the study were from leading brands including Thermo Fisher Scientific (AMPFLSTR IDENTIFILER, AMPFLSTR IDENTIFILER PLUS, GLOBALFILER, AMPFLSTR Y FILER and AMPFLSTR YFILER PLUS), Promega (POWERPLEX 16HS SYSTEM, POWERPLEX 21 SYSTEM, POWERPLEX FUSION 6C SYSTEM and POWERPLEX Y 23 SYSTEM) and Qiagen (INVESTIGATOR IDPLEX PLUS, and INVESTIGATOR ARGUS X-12 MULTIPLEX KIT) were tested for direct amplification protocol. This novel protocol was also tested using VERIFILER PLUS PCR AMPLIFICATION KIT (Thermo Fisher Scientific) and SURE ID PANGLOBAL HUMAN DNA IDENTIFICATION KIT (Health Gene Technologies) as well newly launched but still not used in routine forensic DNA typing kits. The details of multiplex kits used in this study are mentioned in Table 1. This study was designed with the aim to test and validate the direct amplification protocol using commercially available 4, 5 and 6 dyes chemistry based non-direct multiplex kits of leading brands in forensics.

Table 1 Comparative list of various multiplex kits.
Full size table

Result and discussion

Optimizing parameters for direct PCR amplification

For the pilot study, initially 10 saliva samples were directly amplified using both the recommended full reaction volume of 25 µl and a reaction mixture with reduced volume of 10 µl with the help of PCR using all the tested multiplex kits. The DNA profile obtained from both the reaction volumes was evaluated, considering amplification of all the markers to showcase a full DNA profile, peak heights including inter and intra marker balance, observation of stutter and/or other artifacts. In this preliminary test, all the tested samples produced quality DNA profiles with both the reaction volumes and showed concordance. Our few previous studies have also reported use of 10 µl PCR reaction volume for an efficient reaction product, which further supported the reduction of final PCR reaction volume42,43. Direct amplification protocol was further validated for 10 µl reaction volume PCR reaction as per recommended PCR conditions of the particular multiplex kits. Using the above said direct amplification protocol, all the tested samples were directly amplified using non-direct 4 dyes chemistry based autosomal STR marker multiplex kit POWERPLEX 16HS SYSTEM (now not commonly used in forensics), 5 dyes chemistry based autosomal STR marker multiplex kits AMPFLSTR IDENTIFILER, AMPFLSTR IDENTIFILER PLUS, POWERPLEX 21 SYSTEM and INVESTIGATOR IDPLEX PLUS, 6 dyes chemistry based autosomal STR marker multiplex kits GLOBALFILER, POWERPLEX FUSION 6C SYSTEM, VERIFILER PLUS and SURE ID PANGLOBAL HUMAN DNA IDENTIFICATION KIT; Y-STR kits AMPFLSTR YFILER, POWERPLEX Y 23 SYSTEM and AMPFLSTR YFILER PLUS MULTIPLEX KITS; X-STR kit INVESTIGATOR ARGUS X-12 MULTIPLEX KIT. For all the used kits, standard recommended conditions except final reaction volume were followed. Complete and quality DNA profile could be obtained with all the tested multiplex kits using this protocol.

All the tested samples were directly amplified at all the loci with the all the above mentioned multiplex PCR kits used in this study. However, it should be noted that the performance evaluation by the peak heights between the kits is only an approximation. It is because the same loci are labeled with different fluorescent dyes and have different amplicon sizes in different kits22 and for the proper assessment of DNA profile, the overall quality of the profile using profile quality measures is more important44.

In conventional PCR method, Tris EDTA (TE) buffer is used for the final volume adjustment, which has also been reported to inhibit PCR amplification to some extent19,45. In this study, amplification grade water was used for final volume adjustment, which reduced the chances of reaction inhibition due to TE. In the case of DNA degradation or inhibition, larger/high molecular weight loci (usually more that 300 bp loci) reflect low peak height, higher peak imbalance or allele drop outs. This effect leads to development of a DNA profile with a slope peak height, also termed as SKI slope effect19,46. Noticeably, in our study almost all the DNA profiles obtained were having balanced peak heights without SKI slope effect. Rarely, very few DNA profiles were observed with the SKI slope effect which was due to the degradation of the samples. This was confirmed by the usual automated extraction followed by RT PCR of those few samples (data not shown).

Conventional DNA typing versus direct amplification methods

To determine the difference between the conventional DNA typing (including DNA extraction, quality and quantity check, amplification and Genotyping), and the present method of direct amplification, and to estimate the DNA quantity likely to be present in the direct amplification reaction, 10 saliva samples were used for amplification with both the methods. The mean quantity of DNA was found to be 0.45 ng for all the 10 tested samples and most of the multiplex kits claim to be more sensitive for more than 0.25 ng input DNA47,48. With conventional and direct amplification methods, all the alleles were observed at all the loci of respective multiplex kits and balanced high quality DNA profiles were obtained, thus establishing concordance. Further, all the tested samples of this study were directly amplified using commonly used non-direct multiplex kits used in forensic DNA typing (Table 1). Direct PCR amplification protocol from the reference saliva samples by omitting DNA extraction and quantification steps, which resulted in 80% reduction of the turnaround time (Table 2).

Table 2 Turnaround time for DNA typing process.
Full size table

Assessment of DNA profile quality

To assure the quality of DNA profile, Total Peak Height (TPH), Peak Height Ratio (PHR), inter and intra locus balance (local and global balance) of the obtained DNA profiles were analyzed.

TPH is the sum of peak heights of both the heterozygous alleles in the DNA profile. PHR for heterozygous alleles defines the ratio of lower peak height and higher peak height at the specific marker. This value ranges from 0 to 1, where 1 represents the two identical peaks with equal heights and 0 represents the situation where one of the peaks is not observed which might be because of allele drop out due to PCR inhibition or degraded DNA44.

$$ {\text{PHR}} = \begin{array}{*{20}l} {{\text{Height }}\,{\text{of }}\,{\text{lower }}\,{\text{peak}}} \hfill \\ {{\text{PHR}} = \{ - - - - - - - - - - - {\text{for }}\,{\text{a}}\,{\text{ heterozygous }}\,{\text{allele }}\,{\text{at }}\,{\text{particular }}\,{\text{locus}}} \hfill \\ {{\text{Height}}\,{\text{ of}}\,{\text{ higher}}\,{\text{ peak}}} \hfill \\ {} \hfill \\ \end{array} $$
$$ {\text{PHR}} = {1 }\,{\text{for }}\,{\text{true }}\,{\text{homozygous }}\,{\text{allele }}\,{\text{at }}\,{\text{particular }}\,{\text{locus}} $$

Inter and Intra locus balance was measured and represented in the local balance and global balance respectively, which is mean of TPH for all the tested loci in the multiplex kits.

Overall TPH, PHR and global balance quality parameters of DNA profile were evaluated statistically using one way analysis of variance (ANOVA) non parametric applying Friedman test at 5 percent of significant level and Dunn's Multiple Comparison test using Prism GrapghPad v5 software49.

Autosomal STR multiplex kits

AMPFLSTR IDENTIFILER AND AMPFLSTR IDENTIFILER PLUS PCR AMPLIFICATION KIT

DNA profiles obtained with these multiplex kits were found to be complete and balanced. The average TPH and PHR ranged from 6553 to 7833 and from 0.875 to 0.984, respectively for both the kits. Out of 103 DNA profiles, only five DNA profiles were observed with ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles showed no significant variation in the global balance (Figs. 2, 3), (Supplementary Tables S1, S2).

Figure 2
figure 2

Quality assessment of DNA profile by Dunn's multiple comparison test for the AMPFLSTR IDENTIFILER PCR AMPLIFICATION KIT (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image
Figure 3
figure 3

Quality assessment of DNA profile by Dunn's multiple comparison test for the AMPFLSTR IDENTIFILER PLUS PCR AMPLIFICATION KIT (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image

POWERPLEX 16HS SYSTEM

DNA profiles obtained with this multiplex kit was also found to be balanced with an average TPH and PHR ranging from 6593 to 7889 and 0.863 to 0.993 respectively. Seven DNA profiles were observed with ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles exhibited no significant variation in the global balance (Fig. 4), (Supplementary Table S3).

Figure 4
figure 4

Quality assessment of DNA profile by Dunn's multiple comparison test for the POWERPLEX 16HS SYSTEM (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image

POWERPLEX 21 SYSTEM

DNA profiles obtained with this multiplex kit were found to have average TPH and PHR ranging from 6548 to 7925 and from 0.865 to 0.993 respectively. Out of the total studied DNA profiles, ten DNA profiles were observed to have ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles accounted no significant variation in the global balance (Fig. 5), (Supplementary Table S4).

Figure 5
figure 5

Quality assessment of DNA profile by Dunn's multiple comparison test for the POWERPLEX 21 SYSTEM (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image

INVESTIGATOR IDPLEX PLUS KIT

DNA profiles obtained with this multiplex kit were found to have an average TPH and PHR ranging from 6593 to 7883 and from 0.866 to 0.993, respectively. Out of the total studied DNA profiles, fourteen DNA profiles were observed with ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles exhibited no significant variation in the global balance (Fig. 6), (Supplementary Table S5).

Figure 6
figure 6

Quality assessment of DNA profile by Dunn's multiple comparison test for the INVESTIGATOR IDPLEX PLUS KIT (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image

POWERPLEX FUSION 6C SYSTEM

DNA profiles obtained with this multiplex kit were found to have an average TPH and PHR values ranging from 6548 to 7927 and from 0.864 to 0.994, respectively. Out of the total studied DNA profiles, ten DNA profiles were observed with ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles accounted no significant variation in the global balance (Fig. 7), (Supplementary Table S6).

Figure 7
figure 7

Quality assessment of DNA profile by Dunn's multiple comparison test for the POWERPLEX FUSION 6C SYSTEM (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image

GLOBALFILER PCR AMPLIFICATION KIT

DNA profiles obtained with this multiplex kit showed average TPH and PHR values ranging from 6546 to 7924 and from 0.861 to 0.994, respectively. Out of the total studied DNA profiles, ten DNA profiles showed ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles exhibited no significant variation in the global balance (Fig. 8), (Supplementary Table S1).

Figure 8
figure 8

Quality assessment of DNA profile by Dunn's multiple comparison test for the GLOBALFILER PCR AMPLIFICATION KIT (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image

VERIFILER PLUS PCR AMPLIFICATION KIT

DNA profiles obtained with this multiplex kit had an average TPH and PHR values ranging from 6549 to 7928 and from 0.867 to 0.995 respectively. Out of the total studied DNA profiles, nine DNA profiles were with ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles exhibited no significant variation in the global balance (Fig. 9), (Supplementary Table S8).

Figure 9
figure 9

Quality assessment of DNA profile by Dunn's multiple comparison test for the VERIFILER PLUS PCR AMPLIFICATION KIT (A) Peak height ratio (B) Total peak height (C) Global balance.

Full size image

SURE ID PANGLOBAL HUMAN DNA IDENTIFICATION KIT

DNA profiles obtained with this multiplex kit were found to have an average TPH and PHR values ranging from 6556 to 7922 and from 0.866 to 0.996 respectively. Out of the total studied DNA profiles, fifteen DNA profiles showed ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles did not exhibit any significant variation in the global balance (Fig. 10), (Supplementary Table S9).

Figure 10
figure 10

Quality assessment of DNA profile by Dunn's multiple comparison test for the SURE ID PANGLOBAL HUMAN DNA IDENTIFICATION KIT (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image

Y-STR multiplex kits

All the male samples in this study were directly amplified using Y STR multiplex kits. The haplotype data of these samples were statistically evaluated on TPH and global balance parameters. The obtained profiles matched the standard criterion of profile quality index for most of the tested sample.

POWERPLEX Y 23 SYSTEM

DNA profiles obtained with this multiplex kit showed an average TPH range from 3224 to 4019. Out of the total studied DNA profiles, six DNA profiles were observed with ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles accounted no significant variation in the global balance (Fig. 11), (Supplementary Table S10).

Figure 11
figure 11

Quality assessment of DNA profile by Dunn's multiple comparison test for the POWERPLEX Y 23 SYSTEM (A) Total peak height (B) Global balance.

Full size image

AMPFLSTR YFILER PCR AMPLIFICATION KIT

DNA profiles obtained with this multiplex kit were found to have an average TPH values ranging from 3211 to 4015. Out of the total studied DNA profiles, eleven DNA profiles were observed with ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles did not show any significant variation in the global balance (Fig. 12), (Supplementary Table S11).

Figure 12
figure 12

Quality assessment of DNA profile by Dunn's multiple comparison test for the AMPFLSTR YFILER PCR AMPLIFICATION KIT (A) Total peak height (B) Global balance.

Full size image

AMPFLSTR YFILER PLUS PCR AMPLIFICATION KIT

DNA profiles obtained with this multiplex kit were found to have an average TPH ranging from 3213 to 4051. Out of the total studied DNA profiles, seven DNA profiles had ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles showed no significant variation in the global balance (Fig. 13), (Supplementary Table S12).

Figure 13
figure 13

Quality assessment of DNA profile by Dunn's multiple comparison test for the AMPFLSTR YFILER PLUS PCR AMPLIFICATION KIT (A) Total peak height (B) Global balance.

Full size image

X-STR multiplex kit

Since male sample possesses only one X chromosome, it resulted in single peak at particular loci of X-STR marker, and female samples showed two peaks at particular loci due to the two chromosomes. Thus, the DNA profile obtained from the male samples were evaluated for TPH and global balance and the DNA profile obtained from the female samples were evaluated for TPH, PHR and global balance as well. Here we used the mean value of THP, PHR for this evaluation. Conclusive outcomes have been represented in Fig. 14 and Supplementary Table S13 for both the male and female DNA profiles accordingly.

Figure 14
figure 14

Quality assessment of DNA profile by Dunn's multiple comparison test for the INVESTIGATOR ARGUS X-12 MULTIPLEX KIT (A) Total peak height (B) Peak height ratio (C) Global balance.

Full size image

INVESTIGATOR ARGUS X-12 MULTIPLEX KIT

DNA profiles obtained with this multiplex kit were found to have an average TPH value ranging from 6593 to 7949 (for both the male and female DNA profiles) and the mean PHR ranged from 0.847 to 0.999 (for female DNA profiles). Out of the total studied DNA profiles, twenty three DNA profiles were observed with ski slope effect. In Dunn's Multiple Comparison test, at 5 percent significant level, all the DNA profiles showed no significant variation in the global balance (Fig. 14), (Supplementary Table S13).

Overall data suggests that this novel protocol of direct amplification worked well with all the multiplex kits used in this study. The proposed method will not only save the effective cost, but will also curb down the turnaround time. Moreover, this novel direct amplification protocol will supposedly be useful for the speedy analysis of forensic DNA cases and ultimately will lead to the justice dissemination at the earliest.

Quality control

All the authors have passed proficiency test and quality control exercise for DNA fingerprinting from GITAD, Spain (http://gitad.ugr.es/principal.htm). Also internal laboratory control standards were followed and controls provided with multiplex kits were used.

Methods

Sample collection

103 saliva samples from anonymous donors, (50 males and 53 females) who hailed from the Ratlam district of Madhya Pradesh were collected in pre-sterile collection tubes. Written informed consent was taken from the subjects in compliance with the Declaration of Helsinki50. The above said population has already been depicted in our earlier publication, in which the authors typed the same samples for 12 XSTRs41 and 15 autosomal STRs51. All methods were carried out in accordance with the relevant guidelines and regulations. All the experimental protocols were approved by the Institutional committee of DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, MP, India. The samples were brought to the laboratory at 4 °C and were stored at − 20 °C. Saliva samples processed with direct amplification protocol were kept in the laboratory at − 20 °C until final processing. A maximum of one month old samples were processed for the direct amplification.

Polymerase chain reaction (PCR) and capillary electrophoresis (CE) conditions

103 saliva samples were used directly for PCR using the following multiplex systems to test the efficacy of commonly used multiplex systems viz. AMPFLSTR IDENTIFILER, AMPFLSTR IDENTIFILER PLUS, POWERPLEX 21 SYSTEM, INVESTIGATOR IDPLEX PLUS, GLOBALFILER, POWERPLEX FUSION 6C SYSTEM, VERIFILER PLUS, SURE ID PANGLOBAL HUMAN DNA IDENTIFICATION KIT, AMPFLSTR YFILER, POWERPLEX Y 23 SYSTEM, AMPFLSTR YFILER PLUS and INVESTIGATOR ARGUS X-12 MULTIPLEX KITS available for forensic and population genetic purpose. The modifications in the reaction volume, cycle number, and input saliva samples for the respective kits have been shown in Tables 3 and 4.

Table 3 Validated parameters of novel direct amplification protocol.
Full size table
Table 4 Details of recommended and reduced PCR reaction volume.
Full size table

Capillary electrophoresis

In all the cases, 0.3 µl of the total PCR product was run on an Applied Biosystems 3100/3500XL Genetic Analyzer using standard parameters and with the size standards/allelic ladder provided with the respective non-direct multiplex kit and a 10 seconds injection time. The obtained data was analyzed using GeneMapper V3.5/GeneMapper ID version 1.4 softwares (Applied Biosystems, Foster City, CA). All the amplifications were performed using ABI thermal cycler 9700 (Applied Biosystems, Foster City, CA), with the modifications (as mentioned with the respective multiplex kits). Alleles were designated on the basis of number of allele repeats with the help of allelic ladder provided along with the respective multiplex kits. Peak detection threshold was set to 50 RFUs for the allele designation.

Statistical evaluation

To evaluate the quality of DNA profile, the Total Peak Height (TPH), Peak Height Ratio (PHR) and global balance parameters were considered. The overall DNA profile quality of direct amplification method was statistically examined applying ANOVA non parametric by Friedman test at 5 percent of significant level Dunn's Multiple Comparison test using Prism GrapghPad v5 software49.