- Email: [email protected]
Parallel sequencing of 48 Y-chromosome STR and SNP markers
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
Parallel sequencing of 48 Y-chromosome STR and SNP markers Ruiyang Taoa,b, Wei Songc, Ruocheng Xiab,d, Jingyi Zhangb,e, Chong Chenb,f, Suhua Zhangb,*, Chengtao Lia,b,* a
Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, PR China Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of Justice, Shanghai 200063, PR China Minhang Criminal Police, Shanghai Public Security Bureau, Shanghai 200199, PR China d Department of Forensic Medicine, School of Basic Medical Science, Wenzhou Medical University, Wenzhou 325035, PR China e Department of Forensic Science, Medical School of Soochow University, Suzhou 215123, PR China f College of Medicine and Forensics, Xi’an Jiaotong University Health Science Center, Xi’an, 710061 PR China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Massively parallel sequencing (MPS) MiSeq FGx™ forensic genomics system Y chromosome Short tandem repeats (STRs) Single nucleotide polymorphisms (SNPs)
Over the last decade, massively parallel sequencing (MPS) has been rapidly developed, bringing many advantages to forensic genomics. It could simultaneously target multiple genetic markers including short tandem repeats (STRs), insertion and deletion polymorphisms (InDels), and single nucleotide polymorphisms (SNPs). In this study, we developed an assay which can simultaneously sequence 22 Y-STRs and 26 Y-SNPs on the MiSeq FGx™ Forensic Genomics System (Illumina, Inc., San Diego, USA). The primers of the markers were designed using the online DesignStudio (Illumina). Subsequently, DNA libraries were established with the TruSeq Amplicon method. For the pilot study, we sequenced 25 unrelated Chinese Han males. A total of 125 sequencebased alleles were observed at the 22 Y-STR loci, with the average depth of coverage (DoC) ranging from 314.9 x to 637.7 × . For the concordant study, the sequencing results of these samples were fully consistent with genotypes called via capillary electrophoresis (CE). For the SNPs, a preferable performance was observed with the average DoC of 836.5 × . However, inter-locus imbalance was observed at some of the markers, which needs substantial improvement of the primer pool. In general, this customized Y-chromosome STR and SNP panel on the MiSeqFGx™ Forensic Genomics System can provide an effective solution for these markers on Y chromosome, which may allow more forensically relevant information to be obtained from limited forensic materials, such as those from sexual assault and rape cases. Besides, it could also facilitate the forensic practices including paternity testing, genetic determination of pedigree and inter-population migration integration.
1. Introduction Massively parallel sequencing (MPS), also known as next-generation sequencing (NGS) or second-generation sequencing (SGS), has been widely used to many biological subjects since its initial introduction more than a decade ago [1]. It could simultaneously target multiple genetic markers regardless of their types or amplicon sizes, including short tandem repeats (STRs), single nucleotide polymorphisms (SNPs) and insertion and deletion polymorphisms (InDels). Advanced MPS technology has made great progress in sequencing speed, read length and throughput, prevailing over the limitations of traditional capillary electrophoresis (CE)-based technology and bringing many advantages to forensic community. Due to the paternally inherited and male-specific characteristics, the
genetic markers on Y chromosome are usually applied for evolutionary anthropology, judicial forensics (such as sexual assault cases), paternity testing and the genetic genealogy [2,3]. In order to increase the efficiency of Y-chromosome markers for forensic practice, we designed a MPS panel that covers 22 Y-STRs (DYS393, DYS505, DYS456, DYS570, DYS576, DYS522, DYS449, DYS481, DYS19, DYS391, DYS635, DYS439, DYS389-I/-II, DYS612, DYS390, DYS643, DYS533, Y-GATAH4, DYS385b, DYS385a, DYS549, DYS392) and 26 Y-SNPs (rs16980601, rs9786043, rs17269816, rs17316592, rs17276358, rs9786247, rs11096432, rs9786401, rs11096433, rs3848982, rs9306845, rs9786479, rs17276345, rs2075640, rs200634940, rs2032645, rs2032650, rs16980426, rs17323322, rs78149062, rs13447354, rs2032652, rs9786707, rs3906, rs16980711, rs3900).
⁎ Corresponding author at: Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of Justice, Shanghai 200063, PR China. E-mail addresses: [email protected] (S. Zhang), [email protected] (C. Li).
https://doi.org/10.1016/j.fsigss.2019.10.006 Received 27 August 2019; Received in revised form 1 October 2019; Accepted 2 October 2019 1875-1768/ © 2019 Published by Elsevier B.V.
Please cite this article as: Ruiyang Tao, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.006
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
R. Tao, et al.
2. Materials and methods
Custom Amplicon Low Input Kit, 10 ng input DNA is suitable for library preparation. In sensitivity study, the average DoC per dilution (from 10 ng to 2 ng) of 9948 ranged from 548.5 x to 110.3 x for STRs and from 956.0 x to 174.8 x for SNPs. When input DNA less than 2 ng, insufficient DoC, intralocus imbalance and allelic drop-out occurred.
Coordinate positions of the 48 Y-chromosome genetic markers referred to Homo sapiens hg19 genome were submitted to the online DesignStudio (Illumina) for multi-primer designing. Detailed information of the 48 markers are listed in the Supplementary Table S1. All expected PCR amplicons in the multiplex showed a size range within 275 bp. 25 peripheral blood samples were collected from unrelated Chinese Han male individuals with written informed consent according to the protocols approved by the Ethics Committee of the Academy of Forensic Sciences, Ministry of Justice, P.R. China. Genomic DNA (gDNA) were extracted and quantified as described in [4]. DNA libraries were prepared using the TruSeq Custom Amplicon Low Input Kit (Illumina) according to the manufacturer’s protocol. After quality control of DNA libraries, they were mixed and denatured to sequence using MiSeq FGx™ Forensic Genomics System with MiSeq v2 Reagent kit and accordingly sequencing chip (Illumina). The raw sequencing data were extracted as fastq files and used our customized pipelines for sequence analysis. For the concordance study of STRs, 17 Y-STRs were genotyped using the AmpFISTR Yfiler Plus System (Thermo Fisher Scientific, MA, USA) and the rest five Y-STRs were genotyped using our customized Ychromosome CE panel. In the sensitivity testing, Control DNA 9948 (Promega, WI, USA) was amplified with serial dilutions of 10 ng, 5 ng, 2 ng, 1 ng, 500 pg, 250 pg, 125 pg, 62.5 pg, 31.25 pg and 15.125 pg for library preparation.
4. Conclusions A multiplex MPS assay of 22 Y-STRs and 26 Y-SNPs was successfully developed based on the MiSeq FGx™ Forensic Genomics System. The results illustrated this newly established assay provides useful sequence information, which is an effective way to increase the efficiency of STR and SNP markers on Y chromosome for forensic application. While, the primer pool needs improvement to achieve a more balanced performance; further validation studies with larger sample size are also necessary. Declaration of Competing Interest None. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (81625013 and 81772028), the Shanghai Outstanding Academic Leaders Plan (2017485), and the Shanghai Talent Development Funding (2017115). The funding institutions had no role in study design, data analysis, publishing decisions, or manuscript preparation.
3. Results and discussion
Appendix A. Supplementary data
The 25 male individual DNA samples were successfully sequenced with reads ranging from 21,349 x to 50,825 × . The average depth of coverage (DoC) were 460.18 x and 836.5 x for STR and SNP loci, respectively. The average allele ratio (AR) was 72.42% for STR markers, with the lowest (61.25%) observed for DYS612 and the highest (88.68%) observed for DYS533. The average DoC of 22 Y-STRs ranged from 637.7 x (DYS390) to 314.9 x (DYS392), which showed inter-locus imbalance to some extent. Comparing with the genotypes via CE detection, completely concordant results were observed from our MPS assay. A total of 125 sequence-based alleles were found at the 22 YSTRs, versus 113 length-based alleles detected by CE. As a result, haplotype diversity (HD) and discrimination capacity (DC) of these markers increased accordingly with more alleles recognized by sequencing. The separation of alleles with the same length could also facilitate interpret mixed DNA samples. For the 26 SNPs, a preferable performance was evaluated by the parameter adopted in [5]. The average DoC of these SNPs ranged from1318.02 x (rs9306845) to 271.24 x (rs2032652). As recommended in the protocol of TruSeq
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigss.2019.10.006. References [1] S. Goodwin, J.D. McPherson, W.R. McCombie, Coming of age: ten years of nextgeneration sequencing technologies, J. Nat. Rev. Genet. 17 (6) (2016) 333–351. [2] C. Batini, M.A. Jobling, Detecting past male-mediated expansions using the Y chromosome, J. Hum. Genet. 136 (2017) 547–557. [3] K.N. Ballantyne, M. Goedbloed, R. Fang, et al., Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications, J. Am. J Hum. Genet 87 (2010) 341–353. [4] H. Zheng, R. Tao, J. Zhang, et al., Development and validation of a novel SiFaSTRTM 23-plex system, J. Electrophoresis (2019). [5] S. Zhang, Y. Bian, A. Chen, et al., Developmental validation of a custom panel including 273 SNPs for forensic application using Ion Torrent PGM, J. Forensic Sci. Int. Genet. 27 (2017) 50–57.
2
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Parallel sequencing of 48 Y-chromosome STR and SNP markers Ruiyang Taoa,b, Wei Songc, Ruocheng Xiab,d, Jingyi Zhangb,e, Chong Chenb,f, Suhua Zhangb,*, Chengtao Lia,b,* a
Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, PR China Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of Justice, Shanghai 200063, PR China Minhang Criminal Police, Shanghai Public Security Bureau, Shanghai 200199, PR China d Department of Forensic Medicine, School of Basic Medical Science, Wenzhou Medical University, Wenzhou 325035, PR China e Department of Forensic Science, Medical School of Soochow University, Suzhou 215123, PR China f College of Medicine and Forensics, Xi’an Jiaotong University Health Science Center, Xi’an, 710061 PR China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Massively parallel sequencing (MPS) MiSeq FGx™ forensic genomics system Y chromosome Short tandem repeats (STRs) Single nucleotide polymorphisms (SNPs)
Over the last decade, massively parallel sequencing (MPS) has been rapidly developed, bringing many advantages to forensic genomics. It could simultaneously target multiple genetic markers including short tandem repeats (STRs), insertion and deletion polymorphisms (InDels), and single nucleotide polymorphisms (SNPs). In this study, we developed an assay which can simultaneously sequence 22 Y-STRs and 26 Y-SNPs on the MiSeq FGx™ Forensic Genomics System (Illumina, Inc., San Diego, USA). The primers of the markers were designed using the online DesignStudio (Illumina). Subsequently, DNA libraries were established with the TruSeq Amplicon method. For the pilot study, we sequenced 25 unrelated Chinese Han males. A total of 125 sequencebased alleles were observed at the 22 Y-STR loci, with the average depth of coverage (DoC) ranging from 314.9 x to 637.7 × . For the concordant study, the sequencing results of these samples were fully consistent with genotypes called via capillary electrophoresis (CE). For the SNPs, a preferable performance was observed with the average DoC of 836.5 × . However, inter-locus imbalance was observed at some of the markers, which needs substantial improvement of the primer pool. In general, this customized Y-chromosome STR and SNP panel on the MiSeqFGx™ Forensic Genomics System can provide an effective solution for these markers on Y chromosome, which may allow more forensically relevant information to be obtained from limited forensic materials, such as those from sexual assault and rape cases. Besides, it could also facilitate the forensic practices including paternity testing, genetic determination of pedigree and inter-population migration integration.
1. Introduction Massively parallel sequencing (MPS), also known as next-generation sequencing (NGS) or second-generation sequencing (SGS), has been widely used to many biological subjects since its initial introduction more than a decade ago [1]. It could simultaneously target multiple genetic markers regardless of their types or amplicon sizes, including short tandem repeats (STRs), single nucleotide polymorphisms (SNPs) and insertion and deletion polymorphisms (InDels). Advanced MPS technology has made great progress in sequencing speed, read length and throughput, prevailing over the limitations of traditional capillary electrophoresis (CE)-based technology and bringing many advantages to forensic community. Due to the paternally inherited and male-specific characteristics, the
genetic markers on Y chromosome are usually applied for evolutionary anthropology, judicial forensics (such as sexual assault cases), paternity testing and the genetic genealogy [2,3]. In order to increase the efficiency of Y-chromosome markers for forensic practice, we designed a MPS panel that covers 22 Y-STRs (DYS393, DYS505, DYS456, DYS570, DYS576, DYS522, DYS449, DYS481, DYS19, DYS391, DYS635, DYS439, DYS389-I/-II, DYS612, DYS390, DYS643, DYS533, Y-GATAH4, DYS385b, DYS385a, DYS549, DYS392) and 26 Y-SNPs (rs16980601, rs9786043, rs17269816, rs17316592, rs17276358, rs9786247, rs11096432, rs9786401, rs11096433, rs3848982, rs9306845, rs9786479, rs17276345, rs2075640, rs200634940, rs2032645, rs2032650, rs16980426, rs17323322, rs78149062, rs13447354, rs2032652, rs9786707, rs3906, rs16980711, rs3900).
⁎ Corresponding author at: Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of Justice, Shanghai 200063, PR China. E-mail addresses: [email protected] (S. Zhang), [email protected] (C. Li).
https://doi.org/10.1016/j.fsigss.2019.10.006 Received 27 August 2019; Received in revised form 1 October 2019; Accepted 2 October 2019 1875-1768/ © 2019 Published by Elsevier B.V.
Please cite this article as: Ruiyang Tao, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.006
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
R. Tao, et al.
2. Materials and methods
Custom Amplicon Low Input Kit, 10 ng input DNA is suitable for library preparation. In sensitivity study, the average DoC per dilution (from 10 ng to 2 ng) of 9948 ranged from 548.5 x to 110.3 x for STRs and from 956.0 x to 174.8 x for SNPs. When input DNA less than 2 ng, insufficient DoC, intralocus imbalance and allelic drop-out occurred.
Coordinate positions of the 48 Y-chromosome genetic markers referred to Homo sapiens hg19 genome were submitted to the online DesignStudio (Illumina) for multi-primer designing. Detailed information of the 48 markers are listed in the Supplementary Table S1. All expected PCR amplicons in the multiplex showed a size range within 275 bp. 25 peripheral blood samples were collected from unrelated Chinese Han male individuals with written informed consent according to the protocols approved by the Ethics Committee of the Academy of Forensic Sciences, Ministry of Justice, P.R. China. Genomic DNA (gDNA) were extracted and quantified as described in [4]. DNA libraries were prepared using the TruSeq Custom Amplicon Low Input Kit (Illumina) according to the manufacturer’s protocol. After quality control of DNA libraries, they were mixed and denatured to sequence using MiSeq FGx™ Forensic Genomics System with MiSeq v2 Reagent kit and accordingly sequencing chip (Illumina). The raw sequencing data were extracted as fastq files and used our customized pipelines for sequence analysis. For the concordance study of STRs, 17 Y-STRs were genotyped using the AmpFISTR Yfiler Plus System (Thermo Fisher Scientific, MA, USA) and the rest five Y-STRs were genotyped using our customized Ychromosome CE panel. In the sensitivity testing, Control DNA 9948 (Promega, WI, USA) was amplified with serial dilutions of 10 ng, 5 ng, 2 ng, 1 ng, 500 pg, 250 pg, 125 pg, 62.5 pg, 31.25 pg and 15.125 pg for library preparation.
4. Conclusions A multiplex MPS assay of 22 Y-STRs and 26 Y-SNPs was successfully developed based on the MiSeq FGx™ Forensic Genomics System. The results illustrated this newly established assay provides useful sequence information, which is an effective way to increase the efficiency of STR and SNP markers on Y chromosome for forensic application. While, the primer pool needs improvement to achieve a more balanced performance; further validation studies with larger sample size are also necessary. Declaration of Competing Interest None. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (81625013 and 81772028), the Shanghai Outstanding Academic Leaders Plan (2017485), and the Shanghai Talent Development Funding (2017115). The funding institutions had no role in study design, data analysis, publishing decisions, or manuscript preparation.
3. Results and discussion
Appendix A. Supplementary data
The 25 male individual DNA samples were successfully sequenced with reads ranging from 21,349 x to 50,825 × . The average depth of coverage (DoC) were 460.18 x and 836.5 x for STR and SNP loci, respectively. The average allele ratio (AR) was 72.42% for STR markers, with the lowest (61.25%) observed for DYS612 and the highest (88.68%) observed for DYS533. The average DoC of 22 Y-STRs ranged from 637.7 x (DYS390) to 314.9 x (DYS392), which showed inter-locus imbalance to some extent. Comparing with the genotypes via CE detection, completely concordant results were observed from our MPS assay. A total of 125 sequence-based alleles were found at the 22 YSTRs, versus 113 length-based alleles detected by CE. As a result, haplotype diversity (HD) and discrimination capacity (DC) of these markers increased accordingly with more alleles recognized by sequencing. The separation of alleles with the same length could also facilitate interpret mixed DNA samples. For the 26 SNPs, a preferable performance was evaluated by the parameter adopted in [5]. The average DoC of these SNPs ranged from1318.02 x (rs9306845) to 271.24 x (rs2032652). As recommended in the protocol of TruSeq
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigss.2019.10.006. References [1] S. Goodwin, J.D. McPherson, W.R. McCombie, Coming of age: ten years of nextgeneration sequencing technologies, J. Nat. Rev. Genet. 17 (6) (2016) 333–351. [2] C. Batini, M.A. Jobling, Detecting past male-mediated expansions using the Y chromosome, J. Hum. Genet. 136 (2017) 547–557. [3] K.N. Ballantyne, M. Goedbloed, R. Fang, et al., Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications, J. Am. J Hum. Genet 87 (2010) 341–353. [4] H. Zheng, R. Tao, J. Zhang, et al., Development and validation of a novel SiFaSTRTM 23-plex system, J. Electrophoresis (2019). [5] S. Zhang, Y. Bian, A. Chen, et al., Developmental validation of a custom panel including 273 SNPs for forensic application using Ion Torrent PGM, J. Forensic Sci. Int. Genet. 27 (2017) 50–57.
2