| Objective: While the research of mixture has made great progress,there are many problems that have not been effectively resolved and it is still a great challenge in forensic genetics.Due to the limitation of traditional capillary electrophoresis(CE)technology,it is difficult to obtain more genetic information contained in mixture adequately.In recent years,forensic scientists are greatly attracted by next generation sequencing(NGS)technology and tend to apply NGS in forensic cases.Using NGS to detect short tandem repeat(STR)has great advantages than CE,such as obtaining the sequence information other than length information of STR marker,obtaining more STR loci genotype in one test.These advantages might benefit for mixed DNA typing.Therefore,in this study,we evaluated the performance of Precision ID Global Filer NGS STR Panel on two-male mixed DNA samples using Ion Torrent PGM? platform.The mixed DNA profiles obtained from NGS were further analyzed using sep DNA software developed by our lab,aiming at supplying more information for the interpretation of mixed DNA.Methods: 1 Sample collection 50 unrelated healthy male individual blood samples from Hebei Province Blood Center were collected and three DNA samples were used as control DNA,including 9948 male DNA,2800 Control DNA and DNA Control 007.2 DNA extraction Relia Prep? Blood g DNA Miniprep System kit was used to extract whole genome DNA from the above samples.The quality of DNA was examined with Nano Q?.3 DNA quantificationThe whole genome DNA of all samples including the DNA standard samples was quantified on the 7500 Real-time PCR System using Quantifiler? Human DNA Quantification Kit.4 Sample preparation 4.1 The part of laboratory evaluation The library of 9948 male DNA was constructed and sequenced in triplicate in repeatability studies.A total of 20 samples were tested,including 9948 male DNA,2800 Control DNA,DNA Control 007 and 17 blood samples from Hebei Province Blood Center in concordance studies.After quantification,the 9948 Male DNA was employed as a series of input DNA(1 ng,500 pg,200 pg,100 pg and 50 pg)in duplicate in sensitivity studies.4.2 The part of mixture According to the quantification results,50 DNA samples were classified and the DNA samples with close concentration were picked up to construct mixed DNA.Four groups of two-male mixed DNA were developed with different mix ratios including 1:1,1:4,1:9 and 1:19.Each sample was detected in duplicate.5 Library preparation Libraries were prepared using 1ng of DNA with the Precision ID Global Filer? NGS STR Panel and Precision ID Library Kit according to the manufacturer's protocol.6 Template preparation Sequencing template was prepared using Ion PGM? Hi?Q? OT2 Kit on Ion One Touch? 2 System according to the manufacturer's protocol.7 Ion PGM? sequencing Samples were sequenced on Ion PGM? System using Ion PGM? Hi?Q? STR Sequencing Kit according to the manufacturer's protocol.8 Sequencing date analysis Sequencing results were analyzed using Ion Torrent Suite? with the HID_STR_Genotyper and Coverage Analysis.9 CE-STR typingConventional STR typing was completed using the Power Plex? Fusion System for all single samples in order to compare profiles observed by NGS-STR.10 Data analysis Firstly,the influence of analytical threshold that confirms alleles and filters noise was assessed.Then informative metrics including sample depth of coverage(Do C),locus average Do C,locus sequence constituent proportion and allele coverage ratio(ACR)were analyzed to evaluate the quality of the date produced.The comprehensive evaluation in laboratory was performed when the assess of repeatability,concordance and sensitivity was completed.For two male-mixed samples detected by NGS,the parameters were evaluated and the mixed DNA profiles was analysed using a mixed DNA software sep DNA developed by our lab.Results: 1 The lab evaluation of Precision ID Global Filer? NGS STR Panel:1.1 Analytical threshold The vast majority of noises were filtered at the default analytical threshold(that was AT=5%)meeting the requirement to some extent.1.2 Date quality The informative metrics of data quality showed as follows: The average Do C across the samples was 2535×;all loci showed an average Do C 2542×;for sequence constituent proportion,%allele was averaged to 90.83%,%stutter was averaged to 4.22% and %noise was averaged to 5.21%;ACR of all the STR loci was averaged to 0.76 and there were two locus(D12S391 and D12ATA63)showing average ACR< 0.6.1.3 Repeatability studies Reproducible results could be obtained and there was no significant difference in %Allele and ACR among the triplicate in repeatability studies.1.4 Concordance studies Both CE-STR and NGS-STR had overall 761 comparable alleles including 758(99.61%)in concordance and 3(0.39%)in discordance at locus D18S51.In addition,the isoalleles were observed in 8 loci including D2S441,D1S1656,D8S1179,D3S1358,v WA,D21S11,D2S1338 and D12S391.1.5 Sensitivity studies Full and concordant profiles were obtained from input DNA as low as 100 pg,and when the input DNA was down to 50 pg,88.68% of alleles could be detected even though more dropout was observed at the default analytical threshold.Though input DNA was down to 100 pg,ACR showed an average of 0.598,presenting well balance.As the input DNA reduced,average ACR for different input DNA dropped from 0.729 to 0.329,while CV increased from 23.95% to 98.21%.2 The research of mixed DNA using Precision ID Global Filer? NGS STR Panel: Eight single DNA samples were assembled to four groups of two-male mixed DNA.Each group consisted of four different mix ratios.A total of 16 mixed samples were examined in duplicate.The number of alleles was counted by rounding an integer.2.1 Distinguishment of isoalleles by sequence information NGS-STR could identify the difference of allelic sequence,which can distinguish the isoalleles.For example,we observed that the minor contributor(14,17)shared the 17 allele with the major contributor(17,18)at locus v WA for one mixed DNA sample with 1:9 ratio.The minor contributor's 17 allele was distinguished from the major contributor's 17 allele by an intra-allelic sequence variant.Another example was occurred at locus D1S1656,the 11 allele of the minor contributor(11,14)overlapped with the stutter of 12 allele from the the major contributor(12,15),which was distinguished by an intra-allelic sequence variant within the 11 allele.2.2 The average locus Do C of mixed samples at different mix ratios The lowest average Do C was 451±130×(FGA)and the highest was 7263±2105×(Amelogenin)at 1:1 ratio.The lowest average Do C was 698±222×(FGA)and the highest was 10636±3520×(TPOX)at 1:4 ratio.The lowest average Do C was 565±223×(D3S4529)and the highest was 9249±1640×(TPOX)at 1:9 ratio.The lowest average Do C was 563±214×(D3S4529)and the highest was 10571±2176×(TPOX)at 1:19 ratio.2.3 The detection of alleles for different mix ratio The total number of alleles obtained at each ratio(1:1,1:4,1:9 and 1:19)was 365,354,309 and 281 corresponding to 0,0,7,and 15 dropout,and the detected rate of alleles was 100%,96.98%,84.66% and 76.99% respectively.The total number of unshared alleles of minor contributor at each ratio(1:1,1:4,1:9 and 1:19)was 134,134,130 and 121,while including 134,124,78 and 49 unshared alleles of minor contributor above the default analytical threshold,and the detected rate were 100%,92.54%,60.00% and 40.50%,respectively.2.4 The evaluation of parameters of mixed DNA typing results 2.4.1 The heterozygote balance ratio(H_b)The average H_b of major contributor with the minimum 0.70 and the maximum 0.78 in 4 mixed ratios indicated a well balance.The average H_b of minor contributor at 1:1 and 1:4 ratios were 0.73 and 0.67,showing also a well balance.However there had been more variation for the H_b of minor contributor even heterozygosity loss at 1:9 and 1:19 ratios,showing an average H_b of 0.54 and 0.45,respectively.2.4.2 The D value and estimated mixture proportion(Mx1)The distribution of D values on the coordinate interval of D<0.2 and locus Do C<4000×,including 82% of D values less than 0.1 and 93% of D values less than 0.15.The vast majority of D values were less than 0.1 at each mix ratio except for 1:1.The Mx1 presented the minimum 0.47 and the maximum 0.49 at 1:1 ratio,the minimum 0.16 and the maximum 0.19 at 1:4 ratio,the minimum 0.08 and the maximum 0.12 at 1:9 ratio,the minimum 0.05 and the maximum 0.07 at 1:1 ratios.As the mix ratios reduced from 1:1 to 1:19,CV increased from 27.21% to 54.19%.2.5 The analysis of separation accuracy rate by sep DNA software 2.5.1 The overall separation accuracy rate In Best genotype,the overall separation accuracy rates of Bayes model and Iter model were 64.91% and 64.28% respectively,and there was no significant difference between two models(P=0.7734).After considering both Best and Better genotypes,the overall separation accuracy rates of Bayes model and Iter model were 74.18% and 71.13% respectively,and there was no significant difference between two models(P=0.1353).2.5.2 The separation accuracy rate at different mix ratio In Best genotype,the separation accuracy rates of Bayes model at 1:1,1:4,1:9 and 1:19 were 52.15%,75.76%,72.40% and 65.05% respectively;the separation accuracy rates of Iter model at 1:1,1:4,1:9 and 1:19 were 50.14%,75.36%,72.39% and 65.07% respectively.After considering both Best and Better genotypes,the separation accuracy rates of Bayes model at 1:1,1:4,1:9 and 1:19 were 65.17%,84.87%,81.73% and 73.20% respectively;the separation accuracy rates of Iter model at 1:1,1:4,1:9 and 1:19 were 58.82%,83.74%,79.10% and 68.08% respectively.2.5.3 The effects of shared alleles on separation accuracy rate Under Bayes model,the separation accuracy rate of shared locus was 60.00% while unshared locus was 71.83% in Best genotype,which was significantly higher than that of shared locus(P=0.0002).After considering both Best and Better genotypes,the separation accuracy rate of shared locus and unshared locus were both increased to 70.81% and 78.93% respectively,and there was significant difference between them(P=0.0048).Under Iter model,the separation accuracy rate of shared locus was 59.64% while unshared locus was 70.81% in Best genotype,which was significantly higher than that of shared locus(P=0.0004).After considering both Best and Better genotypes,the separation accuracy rate of shared locus and unshared locus were both increased to 66.67% and 77.41% respectively,and there was significant difference between them(P=0.0003).Conclusions: 1 The Precision ID Global Filer NGS STR Panel performed on Ion Torrent PGM? demonstrated a well-performance and could be successfully applied to the genotyping of STR.2 NGS-STR could generate more valuable information than CE-STR for mixed DNA analysis such as isoalleles,partial alleles of minor contributors even at 1:19 ratio.It was feasible to analyze mixed DNA profile get from NGS-STR by utilizing allelic reads as the quantitative information parameter when using sep DNA software. |
PDF Full Text Request