- Email: [email protected]
Genotyping microhaplotype markers through massively parallel sequencing
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Genotyping microhaplotype markers through massively parallel sequencing ⁎
Jing Zhua, Peng Chena, Shengqiu Qua, Hui Wangb, Dan Chenb, Weibo Lianga, , Lin Zhanga, a b
⁎
West China School of basic Science and Forensic Medicine, Sichuan University, Chengdu, Sichuan, China Institute of Forensic Science, Chengdu Public Security Bureau, Chengdu, Sichuan, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Microhaplotype Mixture The ratio of two contributors’ depth of coverage (DOCR)
Mixture interpretation is challenging at present. Short tandem repeats (STR) and single nucleotide polymorphism (SNP) are still in mainstream in forensic genetics, but both of them are not good enough for mixture interpretation. Microhaplotype is a potentially polymorphic genetic marker. In this study, DNA mixtures were simulated at different mixing ratio, and microhaplotype markers were used to analyzing the mixtures through massively parallel sequencing (MPS).
1. Introduction
2. Material and methods
Trace samples recovered from the crime scenes may produce DNA mixtures that contain DNAs of more than one contributor. And the particularly challenging aspect of these forensic cases is the deconvolution of mixture. STR and SNP are common forensic markers used in forensic caseworks. However, most SNP markers are bi-allelic, which makes them be not sufficiently informative for mixture interpretation. STR markers contain much more polymorphic information, but stutters produced during the PCR amplification attributed to enzyme slippage also introduce additional difficulties to genotyping by capillary electrophoresis (CE). Microhaplotype marker is a potentially polymorphic forensic marker which is encompassing more than two SNPs with an extent of < 200 bp [1,2]. And no stutter would be produced during the PCR amplification as microhaplotype markers show their polymorphism in different sequencings rather than different repeating units. After the exploration of our team [3], MPS which is efficient and economical is chosen to genotype this new marker. DNAs were extracted from samples collected from crime scene, subsequently amplified through the PCR process. The primers of STR markers tagged with fluorescent dye are incorporated into the amplified products. After analyzed by CE, the fluorescence intensity of post-amplification product is visualized as peak height. So the amount of starting DNA template is relevant to peak height. Likewise, the depth of sequences may be relevant to the amount of starting DNA template. In this study, we simulated DNA mixture models which comprised DNAs of two contributors. Using MPS to genotype the microhaplotype markers of the DNA mixture models, and analyzing the depth of coverage of different contributors.
Data was downloaded from the 1000 genome project after dealt with VCF to PED Converter, and haplotypes and their relevant frequencies were derived by HAPLOVIEW. 11 microhaplotype loci which contained 3–6 SNPs and showed polymorphic in Han population in China were enrolled in this study. DNA was extracted from peripheral blood of 6 volunteers (A–F). The QIAamp® DNA Mini and Blood Mini Kit (Qiagen, Germany) was used for DNA extraction. After DNA quantification by NanoDrop 1000 (Thermo Fisher, USA), three couples of DNA (A and B, C and D, E and F) were used to simulated a total of 15 DNA mixture models which comprised DNAs of two contributors (the ratio were 1:1, 1:9, 9:1, 1:49 and 49:1). Libraries were prepared through Wafergen platform (BGI, China) and sequencing was conducted on Illumina® Hi-Seq 4000 platform. FASTQ data outputted were deciphered and genotyped by the FLfinder [4] designed by our team. Alleles and their relevant sequences depth of the mixture samples were analyzed and compared with the two contributors’ genotypes.
⁎
3. Results and discussion A total of 15 DNA mixtures (1:1 = 3, 1:9 = 6, and 1:49 = 6) got genotypes of 11 loci. After comparing genotypes of the two contributors which constituted the DNA mixtures with final genotypes of the DNA mixtures, we found some mixtures failed to recover all the alleles at certain loci. The details were in Fig. 1. Six groups of mixture samples (each group contained samples at ratio of 1:1 = 1, 1:9 = 2, and 1:49 = 2) in four loci could be distinctly disentangle, because the two contributors shared no alleles. The ratio of two contributors’ depth of coverage (DOCR) is used as a main
Corresponding authors. E-mail addresses: [email protected] (W. Liang), [email protected] (L. Zhang).
http://dx.doi.org/10.1016/j.fsigss.2017.09.128 Received 30 August 2017; Accepted 19 September 2017 1875-1768/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Zhu, J., Forensic Science International: Genetics Supplement Series (2017), http://dx.doi.org/10.1016/j.fsigss.2017.09.128
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
J. Zhu et al.
Fig. 1. The white boxes represented the samples which failed to recover all alleles of both contributors. The purple boxes represented the samples which recovered all alleles of both contributors. The 11 columns from left to right represented the 11 loci sequentially.
Fig. 2. ⅰshowed an example of sample that the two contributors shared no alleles. And the DOCR was 1.21 at ratio of 1:1, 2.46 and 4.12 at ratio of 1:9. Only the alleles of major DNA were recovered at ratio of 1:49. ⅱshowed an example of sample that the two contributors shared one allele. And the DOCR was 1.40 at ratio of 1:1, 6.08 and 6.59 at ratio of 1:9. Only the alleles of major DNA were recovered at ratio of 1:49.
9.00 ± 4.85. Fig. 2 illustrated a group of mixture samples at locus L5C2A.
informative metric to compare with the ratio of DNAs. DOCR were calculated by dividing the higher coverage contributor by the lower coverage contributor at that locus. The average DOCR of 6 mixture samples at ratio of 1:1 was 2.06 ± 1.07. Half of the twelve samples at ratio of 1:9 recovered all alleles, and the average DOCR was 10.54 ± 8.71. One allele of contributor C was failed to recover in the mixture C:D = 1:9 at locus L2C5A, and the DOCR was 22.67. Other 5 samples at ratio of 1:9 only recovered the alleles of the major DNA. All 12 samples at ratio of 1:49 only recovered the alleles of the major DNA. Fig. 2 illustrated a group of mixture samples at locus L10C4A. The DOCR of a total of 35 samples in 8 loci could be estimated. The two contributors shared one allele and both showed an individual allele. The DOCRs were estimated by the depth of coverage of their individual alleles. The average DOCR of 13 samples at ratio of 1:1 was 2.42 ± 1.86. The average DOCR of 16 samples at ratio of 1:9 was 4.65 ± 2.26. The average DOCR of 6 samples at ratio of 1:49 was
4. Conclusion Genotyping the microhaplotype markers through MPS is a potential methodology for mixture interpretation. When the minor DNA is more than 1/10 in the mixture, more than half samples could recover all alleles of both individuals. And the ratio of two contributors’ depth of coverage (DOCR) may be used to estimate the ratio of DNA in mixture that comprised DNAs of two contributors. Role of funding This study was supported by grants from the National Natural Science Foundation of China (Nos. 81671871). 2
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
J. Zhu et al.
References
Conflict of interest None.
[1] K.K. Kidd, et al., Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics, Forensic Sci. Int. Genet. 12 (2014) 215–224. [2] K.K. Kidd, et al., Evaluating 130 microhaplotypes across a global set of 83 populations, Forensic Sci. Int. Genet. 29 (2017) 29–37. [3] P. Chen, et al., Microhaplotype identified and performed in genetic investigation using PCR-SSCP, Forensic Sci. Int. Genet. 28 (2017) e1–e7. [4] J. Zhu, et al., FLfinder: a novel software for the microhaplotype marker, Forensic Sci. Int. Genet. Suppl. Ser. 5 (2015) e622–e624.
Acknowledgements This project was conducted by our whole team and supported by all the institutions.
3
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Genotyping microhaplotype markers through massively parallel sequencing ⁎
Jing Zhua, Peng Chena, Shengqiu Qua, Hui Wangb, Dan Chenb, Weibo Lianga, , Lin Zhanga, a b
⁎
West China School of basic Science and Forensic Medicine, Sichuan University, Chengdu, Sichuan, China Institute of Forensic Science, Chengdu Public Security Bureau, Chengdu, Sichuan, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Microhaplotype Mixture The ratio of two contributors’ depth of coverage (DOCR)
Mixture interpretation is challenging at present. Short tandem repeats (STR) and single nucleotide polymorphism (SNP) are still in mainstream in forensic genetics, but both of them are not good enough for mixture interpretation. Microhaplotype is a potentially polymorphic genetic marker. In this study, DNA mixtures were simulated at different mixing ratio, and microhaplotype markers were used to analyzing the mixtures through massively parallel sequencing (MPS).
1. Introduction
2. Material and methods
Trace samples recovered from the crime scenes may produce DNA mixtures that contain DNAs of more than one contributor. And the particularly challenging aspect of these forensic cases is the deconvolution of mixture. STR and SNP are common forensic markers used in forensic caseworks. However, most SNP markers are bi-allelic, which makes them be not sufficiently informative for mixture interpretation. STR markers contain much more polymorphic information, but stutters produced during the PCR amplification attributed to enzyme slippage also introduce additional difficulties to genotyping by capillary electrophoresis (CE). Microhaplotype marker is a potentially polymorphic forensic marker which is encompassing more than two SNPs with an extent of < 200 bp [1,2]. And no stutter would be produced during the PCR amplification as microhaplotype markers show their polymorphism in different sequencings rather than different repeating units. After the exploration of our team [3], MPS which is efficient and economical is chosen to genotype this new marker. DNAs were extracted from samples collected from crime scene, subsequently amplified through the PCR process. The primers of STR markers tagged with fluorescent dye are incorporated into the amplified products. After analyzed by CE, the fluorescence intensity of post-amplification product is visualized as peak height. So the amount of starting DNA template is relevant to peak height. Likewise, the depth of sequences may be relevant to the amount of starting DNA template. In this study, we simulated DNA mixture models which comprised DNAs of two contributors. Using MPS to genotype the microhaplotype markers of the DNA mixture models, and analyzing the depth of coverage of different contributors.
Data was downloaded from the 1000 genome project after dealt with VCF to PED Converter, and haplotypes and their relevant frequencies were derived by HAPLOVIEW. 11 microhaplotype loci which contained 3–6 SNPs and showed polymorphic in Han population in China were enrolled in this study. DNA was extracted from peripheral blood of 6 volunteers (A–F). The QIAamp® DNA Mini and Blood Mini Kit (Qiagen, Germany) was used for DNA extraction. After DNA quantification by NanoDrop 1000 (Thermo Fisher, USA), three couples of DNA (A and B, C and D, E and F) were used to simulated a total of 15 DNA mixture models which comprised DNAs of two contributors (the ratio were 1:1, 1:9, 9:1, 1:49 and 49:1). Libraries were prepared through Wafergen platform (BGI, China) and sequencing was conducted on Illumina® Hi-Seq 4000 platform. FASTQ data outputted were deciphered and genotyped by the FLfinder [4] designed by our team. Alleles and their relevant sequences depth of the mixture samples were analyzed and compared with the two contributors’ genotypes.
⁎
3. Results and discussion A total of 15 DNA mixtures (1:1 = 3, 1:9 = 6, and 1:49 = 6) got genotypes of 11 loci. After comparing genotypes of the two contributors which constituted the DNA mixtures with final genotypes of the DNA mixtures, we found some mixtures failed to recover all the alleles at certain loci. The details were in Fig. 1. Six groups of mixture samples (each group contained samples at ratio of 1:1 = 1, 1:9 = 2, and 1:49 = 2) in four loci could be distinctly disentangle, because the two contributors shared no alleles. The ratio of two contributors’ depth of coverage (DOCR) is used as a main
Corresponding authors. E-mail addresses: [email protected] (W. Liang), [email protected] (L. Zhang).
http://dx.doi.org/10.1016/j.fsigss.2017.09.128 Received 30 August 2017; Accepted 19 September 2017 1875-1768/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Zhu, J., Forensic Science International: Genetics Supplement Series (2017), http://dx.doi.org/10.1016/j.fsigss.2017.09.128
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
J. Zhu et al.
Fig. 1. The white boxes represented the samples which failed to recover all alleles of both contributors. The purple boxes represented the samples which recovered all alleles of both contributors. The 11 columns from left to right represented the 11 loci sequentially.
Fig. 2. ⅰshowed an example of sample that the two contributors shared no alleles. And the DOCR was 1.21 at ratio of 1:1, 2.46 and 4.12 at ratio of 1:9. Only the alleles of major DNA were recovered at ratio of 1:49. ⅱshowed an example of sample that the two contributors shared one allele. And the DOCR was 1.40 at ratio of 1:1, 6.08 and 6.59 at ratio of 1:9. Only the alleles of major DNA were recovered at ratio of 1:49.
9.00 ± 4.85. Fig. 2 illustrated a group of mixture samples at locus L5C2A.
informative metric to compare with the ratio of DNAs. DOCR were calculated by dividing the higher coverage contributor by the lower coverage contributor at that locus. The average DOCR of 6 mixture samples at ratio of 1:1 was 2.06 ± 1.07. Half of the twelve samples at ratio of 1:9 recovered all alleles, and the average DOCR was 10.54 ± 8.71. One allele of contributor C was failed to recover in the mixture C:D = 1:9 at locus L2C5A, and the DOCR was 22.67. Other 5 samples at ratio of 1:9 only recovered the alleles of the major DNA. All 12 samples at ratio of 1:49 only recovered the alleles of the major DNA. Fig. 2 illustrated a group of mixture samples at locus L10C4A. The DOCR of a total of 35 samples in 8 loci could be estimated. The two contributors shared one allele and both showed an individual allele. The DOCRs were estimated by the depth of coverage of their individual alleles. The average DOCR of 13 samples at ratio of 1:1 was 2.42 ± 1.86. The average DOCR of 16 samples at ratio of 1:9 was 4.65 ± 2.26. The average DOCR of 6 samples at ratio of 1:49 was
4. Conclusion Genotyping the microhaplotype markers through MPS is a potential methodology for mixture interpretation. When the minor DNA is more than 1/10 in the mixture, more than half samples could recover all alleles of both individuals. And the ratio of two contributors’ depth of coverage (DOCR) may be used to estimate the ratio of DNA in mixture that comprised DNAs of two contributors. Role of funding This study was supported by grants from the National Natural Science Foundation of China (Nos. 81671871). 2
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
J. Zhu et al.
References
Conflict of interest None.
[1] K.K. Kidd, et al., Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics, Forensic Sci. Int. Genet. 12 (2014) 215–224. [2] K.K. Kidd, et al., Evaluating 130 microhaplotypes across a global set of 83 populations, Forensic Sci. Int. Genet. 29 (2017) 29–37. [3] P. Chen, et al., Microhaplotype identified and performed in genetic investigation using PCR-SSCP, Forensic Sci. Int. Genet. 28 (2017) e1–e7. [4] J. Zhu, et al., FLfinder: a novel software for the microhaplotype marker, Forensic Sci. Int. Genet. Suppl. Ser. 5 (2015) e622–e624.
Acknowledgements This project was conducted by our whole team and supported by all the institutions.
3