New Codis 2021
New Codis 2021
Gene Reports
journal homepage: www.elsevier.com/locate/genrep
A R T I C L E I N F O A B S T R A C T
Keywords: This study presents genetic diversity at 22 autosomal STR loci (extended CODIS STR) in the population of
CODIS STR Rajasthan. It provides an allelic database of this population for the purpose of forensic inference. 595 unrelated
Polymorphism healthy individuals residing in the state were randomly selected from the routine casework of the laboratory.
Genetic database
Compliance of ethical standards was ensured during the collection of blood samples. The allele 15 of locus
Paternity index
D22S1045 was found to be the most frequent allele among all the studied genetic markers. The matching
Rajasthan
probability was found to be 1.80 × 10− 26 at all the studied markers. The loci Penta E and TPOX were found to be
highest and least forensic importance respectively, among all the studied loci. The heterozygosity ranged from
0.711 for locus TPOX to 0.926 for the locus Penta E. The discrimination and exclusion power were found as 1 and
0.999999998292 respectively at all the studied loci. Overall, the highest polymorphism was observed 0.907 for
locus Penta E among the studied markers in the population of Rajasthan. In population differentiation test, the
population of Rajasthan showed a greater genetic affinity with north-western and central Indian populations than
southern and eastern Indian, as well as East Asian populations.
1. Introduction scientific evidence” and has come under public scrutiny. More and more
courts have begun to accept DNA-based evidence. DNA technology has
The state of Rajasthan is located in the North-western region of India taken an unparalleled position in the forensic sciences field for the
(Fig. 1). Its western international boundaries are formed by Punjab and investigation of paternity and criminal cases across the globe (Kumar
Sindh provinces of Pakistan and remaining borders are formed by Indian et al., 2019). Since 1985, when Peter Gill and Alec Jeffrey first applied
states like Haryana, Punjab, Uttar Pradesh, Madhya Pradesh and Gujarat DNA technology to forensic problems, to this day, more than 50,000
(Gupta and Bakshi, 2008) (Fig. S1). Rajasthan was earlier known as cases worldwide have been solved by using DNA-based technology. DNA
“Rajputana” which means “Land of Kings” (Gupta and Bakshi, 2008). Its analysis based on autosomal as well as sex chromosome STR is routinely
gene-pool is known as the oldest one across the globe. According to used in forensic casework, population study and medico-legal in
Census 2011, Rajasthan is inhabited by5.66% of the total population of vestigations. Although the advancement of DNA technology in forensic
India. Demography of Rajasthan comprises of 68,548,437 people science has been extremely rapid, today we are witnessing a new era of
(35,550,997 males and 32,997,440 females) (John, 2011). Migration of DNA technology that uses automation and miniaturization like Rapid
people of Rajasthan is reflected in its genetic diversity (Lawson et al., DNA Typing (Shrivastava et al., 2020).
2012). In Combined DNA Index System (CODIS), short tandem repeat loci
In forensic science, DNA analysis has become “the new form of are required for the forensic DNA Analysis purposes. These core loci
Abbreviations: STR, Short Tendem Repeat; CODIS, Combined DNA Index System; coPD, Combined Power of Discrimination; coPE, Combined Power of Exclusion;
PIC, Polymorphic Information Content; PM, Probability of a Match; HIPS, Human Identification Professional Services; ILS, Internal Lane Standard; Hi Di Formamide,
Highly Deionized Formamide.
* Corresponding author.
E-mail address: [email protected] (A. Kumar).
1
Authors with equal credential.
https://doi.org/10.1016/j.genrep.2021.101150
Received 13 January 2021; Received in revised form 14 March 2021; Accepted 8 April 2021
Available online 15 April 2021
2452-0144/© 2021 Elsevier Inc. All rights reserved.
A. Kumar et al. Gene Reports 23 (2021) 101150
have been expanded to 20 STRs by the use of CODIS (Hares, 2015; 2. Materials and methods
Moretti et al., 2016). Until 01.01.2017, CODIS prescribed 13 core loci
for forensic purposes are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, 2.1. Samples
D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51 and D21S11,
and since 1ST January 2017, seven new markers namely D1S1656, Blood samples of 595 unrelated randomly selected normal adult in
D2S441, D2S1338, D10S1248, D12S391, D19S433 and D22S1045 are dividuals were taken for the study with prior written informed consent
included in the extended CODIS markers. In this study we explore from the candidates. These samples were from the routine casework
genomic diversity on 20 CODIS markers along with two additional Penta performed at DNA division, State Forensic Science Laboratory, Jaipur.
E and Penta D markers in the population of Rajasthan. The allelic di
versity and several forensic parameters for 13 CODIS core loci have been 2.2. DNA isolation and quantification
already reported for the population of Rajasthan (Kumar et al., 2020)
and various states of India (Shrivastava et al., 2015).Therefore, there is DNA was isolated from the blood samples using PrepFiler Express™
utmost need of forensic parameter data based on extended CODIS core kit (Thermo Fisher Scientific, CA, USA-Thermo) with the help of Auto
loci. mate Express (Thermo) according to the recommended protocol of the
This study provides allelic frequencies and various forensic param manufacturers. The quantification of the isolated DNA was performed
eter dataset on extended CODIS core loci for the purpose of forensic by using QuantifilerTrio quantification kit (Thermo) on Quant Studio 5
inferences in the population of Rajasthan. In order to discover genetic (Thermo) instrument as per the prescribed protocol of the producer.
affinity of geographically close Indian populations viz., Central Indian
Population, Uttar Pradesh, Balmiki (Punjab), Jharkhand; geographically 2.3. Amplification and genotyping
distinct Indian populations viz., Mahadev Koli (Maharashtra), Kora
(Bengal), Maheli (Bengal), Tamil Population (Tamil Nadu) and reported Quantified DNA was amplified for 22 extended CODIS core auto
populations of Rajasthan, their pre-existing databases were taken into somal STRs along with gender determining loci Amelogenin and one Y-
consideration. Comparison of genomic ancestry of studied population STR DYS391 using PowerPlex® Fusion system kit (Promega, CA, USA-
with reported global populations was also performed. Promega). Primers for the studied STR markers were presented in
Table 1. Amplification reaction was performed on Veriti™ fast thermal
cycler (Thermo) as per the recommendations of manufacturer except the
2
A. Kumar et al. Gene Reports 23 (2021) 101150
Table 1
Primer sequence of studied STR markers.
Locus Primer sequence [Forward (top row) & Reverse (bottom row)]
Amelogenin 5′ -CCCTGGGCTCTGTAAAGAATAGTG-3′
5′ -ATCAGAGCTTAAACTGGGAAGCTG-3′
D3S1358 5′ -ACT GCA GTC CAA TCT GGG T-3′ (AGAT strand)
5′ -ATG AAA TCA ACA GAG GCT TG-3′ (TCTA strand)
D1S1656 5′ -GTGTTGCTCAAGGGTCAACT-3′
5′ -GAGAAATAGAATCACTAGGGAACC-3′
D2S441 5′ -TGCACCCAACATTCTAACAA-3′
5′ -ATTGGAGCTAAGTGGCTGTG-3′
D10S1248 5′ -GGAATAAGTGCAGTGCTTGG-3′
5′ -ACCAATCTGGTCACAACCAT-3′
D13S317 5′ -ACAGAAGTCTGGGATGTGGA-3′
5′ -GCCCAAAAAGACAGACAGAA-3′
PENTA-E 5′ -ATTACCAACATGAAAGGGTACCAATA-3′
5′ -TGGGTTATTAATTGAGAAAACTCCTTACAATTT-3′
D16S539 5′ -GATCCCAAGCTCTTCCTCTT-3′
5′ -ACGTTTGTGTGTGCATCTGT-3′
D18S51 5′ -TTCTTGAGCCCAGAAGGTTA-3′
5′ -ATTCTACCAGCAACAACACAAATAAAC-3′
D2S1338 5′ -CCAGTGGATTTGGAAACAGA-3′
5′ -ACCTAGCATGGTACCTGCAG-3′
CSF1PO 5′ -AACCTGAGTCTGCCAAGGACTAGC-3′ (AGAT strand)
5′ -TTCCACACACCACTGGCCATCTTC-3′ (TCTA strand)
PENTA-D 5′ -GAAGGTCGAAGCTGAAGTG-3′
5′ -ATTAGAATTCTTTAATCTGGACACAAG-3′
TH01 5′ -ATTCAAAGGGTATCTGGGCTCTGG-3′
5′ -GTGGGCTGAAAAGCTCCCGATTAT-3′
vWA 5′ -GCCCTAGTGGATGATAAGAATAATCAGTATGTG-3′
5′ -GGACAGATGATAAATACATAGGATGGATGG-3′
D21S11 5′ -ATATGTGAGTCAATTCCCCAAG-3′
5′ -TGTATTAGTCAATGTTCTCCAGAGAC-3′
D7S820 5′ -TGTCATAGTTTAGAACGAACTAACG-3′
5′ -CTGAGGTATCAAAAACTCAGAGG-3′
D5S818 5′ -GGGTGATTTTCCTCTTTGGT-3′
5′ -TGATTCCAATCATAGCCACA-3′
TPOX 5′ -GCACAGAACAGGCACTTAGG-3′
5′ -CGCTCAAACGTGAGGTTG-3′
D8S1179 5′ -ATTGCAACTTATATGTATTTTTGTATTTCATG-3′
5′ -ACCAAATTGTGTTCATGAGTATAGTTTC-3′
D12S391 5′ -AACAGGATCAATGGATGCAT-3′
5′ -TGGCTTTTAGACCTGGACTG-3′
D19S433 5′ -ATTTCTAAGGCTGGGTGAGGTG-3′
5′ -TTAAGGAACAGGTGGTGTTGGTT-3′
FGA 5′ -GGCTGCAGGGCATAACATTA-3′
5′ -ATTCTATGACTTTGCGCTTCAGGA-3′
D22S1045 5′ -GCTAGATTTTCCCCGATGAT-3′
5′ -ATGTAAAGTGCTCTCAAGAGTGC-3′
10 μL reaction volume. Some of the samples were directly subjected to (for allele frequency and forensic interest parameters such as, Paternity
amplification as per the previous study (Kumawat et al., 2019)(Kuma Index, discrimination power, exclusion power, estimation of matching
wat et al., 2020, Srivastava et al., 2019). Amplicons were separated on probability, polymorphism, observed and expected heterozygosity);
Genetic Analyzer 3500XL using POP™-4 (Performance Optimized Arlequin V3.5 (for Hardy-Wienberg equilibrium expectations using
Polymer), 36 cm Capillary array as per the recommendations of the exact test); POPTREE2 program and PAST 3.02a (for phylogenetic
manufacturer. Each amplified sample was mixed with 0.5 μL ILS 500 and relatedness between studied and compared populations which were
9.5 μL Hi-Di Formamide. Samples were injected at 1.2 kV for 5 s. Elec evaluated based on Nei’s DA distance through and plotted between
trophoresis results were analyzed with GeneMapper ID-X v1.1 software component 1 and 2); and heat map packages (for heat map).
(Thermo).
3. Results and discussion
2.4. Quality standards
A total of 267 alleles were observed among all the studied loci in the
Our laboratory has been validated by Human Identification Profes population of Rajasthan. The highest number of the alleles were
sional Services (HIPS) by Thermo Fisher Scientific CA, USA. As per the observed at locus Penta E (21) whereas the lowest number of the alleles
internal laboratory standards, positive and negative control samples were found at locus TH01 (6). A wide range of allele frequency with a
were used to ascertain the quality of analysis. minimum value of 0.001 to a maximum value of 0.425 was observed for
the studied population among all the tested loci. Allele 15 of locus
D22S1045 was found to be the most frequent allele among all the
2.5. Statistical analysis
studied genetic markers (Table 2). To find Hardy Weinberg Equilibrium
(HWE) in the allelic data, the exact test was performed; all the loci
Genetic data of present study was statistically evaluated using
followed Hardy Weinberg’s expectations, which supports the random
various population softwares viz., PowerStats V1.2 spreadsheet program
3
A. Kumar et al.
Table 2
Allele frequency observed at autosomal STR loci of PowerPlex® Fusion 5C system in the population of Rajasthan (n = 595)
Allele D3S1358 D1S1656 D2S441 D10S1248 D13S317 PENTA-E D16S539 D18S51 D2S1338 CSF1PO PENTA-D TH01 vWA D21S11 D7S820 D5S818 TPOX D8S1179 D12S391 D19S433 FGA D22S1045
– – – – – – – – – – – – – – – – – – – – –
17.3 – 0.026 – – – – – – – – – – – – – – – – 0.012 – – –
17.4 – – – – – 0.001 – – – – – – – – – – – – – – – –
18 0.112 0.003 0.001 0.005 – 0.050 – 0.034 0.175 – 0.001 – 0.194 – – – – 0.004 0.234 0.001 0.005 0.005
18.3 – 0.015 – – – – – – – – – – – – – – – – 0.017 – – –
19 0.012 – – 0.001 – 0.029 – 0.038 0.173 – – – 0.066 – – – – – 0.144 – 0.045 0.003
19.1 – – – – – – – – – – – – – – – – – – 0.002 – – –
19.2 – – – – – – – – – – – – – – – – – – 0.001 – – –
19.3 – 0.002 – – – – – – – – – – – – – – – – 0.003 – – –
20 – – – – – 0.015 – 0.022 0.110 – – – 0.008 – – – – – 0.107 – 0.115 –
20.2 – – – – – – – – – – – – – – – – – – – – 0.002 –
21 – – – – – 0.008 – 0.009 0.039 – – – 0.001 – – – – – 0.118 – 0.155 –
21.2 – – – – – – – – – – – – – – – – – – – – 0.002 –
22 – – – – – 0.003 – 0.004 0.067 – – – – – – – – – 0.089 – 0.128 –
22.2 – – – – – – – – – – – – – – – – – – – – 0.011 –
23 – – – – – 0.003 – 0.001 0.183 – – – – – – – – – 0.071 – 0.182 –
23.2 – – – – – – – – – – – – – – – – – – – – 0.007 –
24 – – – – – 0.001 – – 0.104 – – – – – – – – – 0.039 – 0.182 –
24.2 – – – – – – – – – – – – – – – – – – – – 0.004 –
25 – – – – – – – – 0.062 – – – – – – – – – 0.011 – 0.115 –
26 – – – – – – – – 0.019 – – – – – – – – – 0.002 – 0.034 –
D22S1045
(Ho) and expected heterozygosity (He) ranged from 0.711 and 0.706 to
0.924 and 0.914 respectively. The observed heterozygosity was found to
be more than 0.7 which is highly supportive of this data for population
–
–
–
–
–
–
–
–
–
–
genetic studies. The allelic data was evaluated for forensic character
FGA
–
–
–
–
–
–
–
–
–
–
content (PIC), power of exclusion (PE), matching probability (Pm), and
D19S433
paternity index (PI). The locus Penta E was found to be most poly
morphic with a value of 0.907 and locus TPOX was found to be least
–
–
–
–
–
–
–
–
–
–
polymorphic with a value of 0.656 among all the studied loci (Table 3).
D12S391
The highest gene diversity (GD) was observed at locus Penta E with a
value of 0.914 and the lowest GD was observed at locus TPOX with a
–
–
–
–
–
–
–
–
–
–
(PE) ranged from 0.858 and 0.445 to 0.984 and 0.845 respectively. The
combined value of the power of discrimination (coPD) and power of
–
–
–
–
–
–
–
–
–
–
of 0.016 and 1.730 to 0.142 and 6.611 respectively (Table 3). The cu
D5S818
0.006
0.001
0.039
0.132
0.004
0.184
0.068
0.002
0.001
0.008
(Kumar et al., 2020, Kumawat et al., 2020) were taken into consider
ation. To know genomically shared ancestry of studied population, re
ported global populations viz., Americans (Basque) (Besecker et al.,
–
–
–
–
–
–
–
–
–
–
well as Tibetan (Gayden et al., 2009) and Korean populations (Yoo et al.,
D18S51
and Manchu population of China (Fig. 2). The distance matrix plotted
using Principal Component 1 and 2 disclosed 77.215% variations. The
–
–
–
–
–
–
–
–
–
–
map (Fig. 4) revealed that the studied population showed greater genetic
D2S441
32.2
33.2
33.3
34.1
34.2
35.2
36.2
Allele
31
32
5
A. Kumar et al. Gene Reports 23 (2021) 101150
Table 3
Forensic parameters for the population of Rajasthan, India (N = 595).
Locus GD PIC PM PD Ho He PE TPI pHW
D3S1358 0.749 0.705 0.105 0.895 0.741 0.748 0.495 1.932 0.406
D1S1656 0.881 0.868 0.026 0.974 0.850 0.880 0.696 3.343 0.950
D2S441 0.719 0.675 0.125 0.875 0.724 0.718 0.467 1.814 0.906
D10S1248 0.774 0.737 0.091 0.909 0.783 0.773 0.568 2.306 0.057
D13S317 0.798 0.769 0.071 0.929 0.775 0.798 0.553 2.220 0.240
PENTA-E 0.914 0.907 0.016 0.984 0.924 0.914 0.845 6.611 0.010
D16S539 0.802 0.775 0.067 0.933 0.812 0.801 0.621 2.656 0.735
D18S51 0.848 0.832 0.041 0.959 0.857 0.848 0.709 3.500 0.394
D2S1338 0.870 0.856 0.033 0.967 0.855 0.870 0.706 3.459 0.040
CSF1PO 0.725 0.678 0.124 0.876 0.711 0.724 0.445 1.730 0.741
PENTA-D 0.827 0.803 0.054 0.946 0.810 0.826 0.618 2.633 0.682
TH01 0.776 0.739 0.087 0.913 0.785 0.775 0.571 2.324 0.691
vWA 0.803 0.775 0.070 0.930 0.829 0.803 0.653 2.917 0.575
D21S11 0.855 0.838 0.039 0.961 0.845 0.855 0.686 3.234 0.383
D7S820 0.800 0.770 0.071 0.929 0.783 0.800 0.568 2.306 0.110
D5S818 0.732 0.687 0.118 0.882 0.724 0.732 0.467 1.814 0.013
TPOX 0.706 0.656 0.142 0.858 0.714 0.706 0.451 1.750 0.557
D8S1179 0.847 0.827 0.044 0.956 0.839 0.846 0.673 3.099 0.451
D12S391 0.866 0.851 0.033 0.967 0.847 0.865 0.689 3.269 0.726
D19S433 0.825 0.803 0.051 0.949 0.820 0.824 0.637 2.780 0.423
FGA 0.864 0.848 0.035 0.965 0.855 0.863 0.706 3.459 0.204
D22S1045 0.721 0.679 0.126 0.874 0.716 0.720 0.453 1.760 0.259
GD- Gene Diversity; PIC- Polymorphic Information Content; Pm – Matching Probability; PD – power of discrimination; Ho - observed heterozygosity; He - expected
heterozygosity; PE- Power of Exclusion; TPI – typical paternity index; pHW - Hardy–Weinberg equilibrium p value.
Fig. 2. Neighbor Joining Tree showing relatedness of 19 compared populations based on 15 autosomal STR data.
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A. Kumar et al. Gene Reports 23 (2021) 101150
Fig. 3. PCA plot indicating the distance and dissimilarity among 19 compared populations based on 15 autosomal STR data.
7
A. Kumar et al. Gene Reports 23 (2021) 101150
4. Conclusion Burman populations in the Himalayas. J. Hum. Genet. 54, 216–223. https://doi.org/
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University, Varanasi, India (Ref. No. - I.Sc./ECM-XII/2018-19/06). Kumar, A., Kumar, R., Kumawat, R.K., Tilawat, A., Shrivastava, P., Chaubey, G., 2020.
Genetic variation (population database) at 20 autosomal STR loci in the population
Funding of Rajasthan (north-western India). Int. J. Legal Med. 1–3.
Kumar, A., Kumar, R., Mohsin, U., Sharma, S., 2019. Genetic Profiling of Short Tandem
Repeat (STR) in forensic science to Crackdown complex cases of sexual abuses.
This research received no external funding. Advances in Bioresearch 10 (5), 29–34. In this issue.
Kumawat, R.K., Shrivastava, Pankaj, Divya, Shrivastava, Mathur, G.K., S., D., 2019.
Genomic blueprint of population of Rajasthan based on autosomal STR markers.
CRediT authorship contribution statement Ann. Hum. Biol. https://doi.org/10.1080/03014460.2019.1705390.
Kumawat, R.K., Shrivastava, P., Shrivastava, D., Mathur, G.K., Dixit, S., 2020. Genomic
AK and RK designed the study, AK analyzed the samples, AK, PS and blueprint of population of Rajasthan based on autosomal STR markers. Ann. Hum.
Biol. 1–6.
RK (Kumawat) did the statistical analysis of the obtained genetic data. Lawson, D.J., Hellenthal, G., Myers, S., Falush, D., 2012. Inference of population
AK and RK (Kumawat) wrote the manuscript. RK, PS and GC reviewed structure using dense haplotype data. PLoS Genet. 8.
the manuscript. All authors read and approved the final manuscript. Liu, J., Guo, L., Qi, R., Li, S., Yin, J. Yang, Zhang, W., Sun, Z., Tian, X., Gao, B., 2013.
Allele frequencies of 19 autosomal STR loci in Manchu population of China with
phylogenetic structure among worldwide populations. Gene 529, 282–287. https://
Declaration of competing interest doi.org/10.1016/j.gene.2013.07.033.
Moretti, T.R., Moreno, L.I., Smerick, J.B., Pignone, M.I., Hizon, R., Buckleton, J.S.,
Bright, J.A., Onorato, A.J., 2016. Population data on the expanded CODIS core loci
The authors declare that they do not have any conflict of interest. for eleven populations of significance for forensic DNA analysis in the United States.
Forensic Sci. Int. Genet. 25, 175–181. In this issue.
Nei, M., 1972. Genetic distance between populations. Am. Nat. 106, 283–292.
Acknowledgment Shrivastava, P., Jain, T., Trivedi, V. Ben, 2015. Genetic polymorphism study at 15
autosomal locus in central Indian population. Springerplus 4, 566.
Authors are thankful to the Director, State Forensic Science Labo Shrivastava, P., Mishra, A., Kumar, A., Choudhary, S., Kakkar, S., Kumawat, R.K., 2020.
Rapid DNA Typing. Forensic DNA Typing; Principles, Applications and
ratory, Rajasthan for permitting the study. Advancements, 1. # Springer-Verlag GmbH Germany, part of Springer Nature 2020,
pp. 561–572. In this issue.
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