Abstract
Researchers have sought to develop an effective protocol for paternity analysis using cell-free DNA (cfDNA) in maternal plasma. The use of massively parallel sequencing (MPS) technology for SNP testing is attractive because of its high-throughput capacity and resolution to single-base precision. In this study, we designed a customized SNP panel for cfDNA sequencing that includes 720 short amplicons (< 140 bp) targeting SNPs on the autosome and Y chromosome. The systemic performance was evaluated using the Ion Torrent PGM, indicating balanced coverage among most of the included loci, except for 78 poorly performing SNPs that were observed to have an inconsistent allele balance, lower coverage reads or high background signals. Then, the custom panel was used to perform cfDNA genotyping in maternal plasma from 20 pregnancies in the first and second trimesters (9 to 21 weeks). By establishing an allele fraction cutoff of 2.0%, 53 to 128 autosomal SNP loci were considered informative for paternal origin. Validation results in foetal samples showed that 49.43% to 100% of the real paternal alleles were accurately identified, with incorrect alleles encountered in 3 cases. The concentration of foetal cfDNA ranged from 4.28% to 10.70%. Our results show that this amplicon-based sequencing strategy could be utilized in analysing paternally inherited alleles in maternal plasma. However, further studies and optimization are required for a more detailed and accurate interpretation of the cfDNA sequencing results based on MPS technology.
Similar content being viewed by others
References
Webb A, Madgett T, Miran T, Sillence K, Kaushik N, Kiernan M, Avent N (2012) Noninvasive prenatal diagnosis of aneuploidy: next generation sequencing or fetal DNA enrichment? Balkan J Med Genet 15(Suppl):17–26
Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350:485–487
Yu SC, Lee SW, Jiang P, Leung TY, Chan KC, Chiu RW, Lo YM (2013) High-resolution profiling of fetal DNA clearance from maternal plasma by massively parallel sequencing. Clin Chem 59(8):1228–1237
Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM (1999) Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 64:218–224
Lo YM, Patel P, Wainscoat JS, Sampietro M, Gillmer MD, Fleming KA (1989) Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 2:1363–1365
Lo YM, Bowell PJ, Selinger M, Mackenzie IZ, Chamberlain P, Gillmer MD, Littlewood TJ, Fleming KA, Wainscoat JS (1993) Prenatal determination of fetal RhD status by analysis of peripheral blood of rhesus negative mothers. Lancet 341:1147–1148
Chiu RW, Chan KC, Gao Y, Lau VY, Zheng W, Leung TY, Foo CH, Xie B, Tsui NB, Lun FM, Zee BC, Lau TK, Cantor CR, Lo YM (2008) Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A 105(51):20458–20463
Barrett AN, McDonnell TC, Chan KC, Chitty LS (2012) Digital PCR analysis of maternal plasma for noninvasive detection of sickle cell anemia. Clin Chem 58:1026–1032
Deng Z, Wu G, Li Q, Zhang X, Liang Y, Li D, Gao S, Lan Y (2006) Noninvasive genotyping of 9 Y-chromosome specific STR loci using circulatory fetal DNA in maternal plasma by multiplex PCR. Prenat Diagn 26:362–368
Wagner J, Dzijan S, Marjanović D, Lauc G (2009) Non-invasive prenatal paternity testing from maternal blood. Int J Legal Med 123:75–79
Barra GB, Santa Rita TH, Chianca CF, Velasco LF, de Sousa CF, Nery LF, Costa SS (2015) Fetal male lineage determination by analysis of Y-chromosome STR haplotype in maternal plasma. Forensic Sci Int Genet 15:105–110
Tynan JA, Mahboubi P, Cagasan LL, van den Boom D, Ehrich M, Oeth P (2011) Restriction enzyme-mediated enhanced detection of circulating cell-free fetal DNA in maternal plasma. J Mol Diagn 13:382–389
Guo X, Bayliss P, Damewood M, Varney J, Ma E, Vallecillo B, Dhallan R (2012) A noninvasive test to determine paternity in pregnancy. N Engl J Med 366:1743–1745
Ryan A, Baner J, Demko Z, Hill M, Sigurjonsson S, Baird ML, Rabinowitz M (2013) Informatics-based, highly accurate, noninvasive prenatal paternity testing. Genet Med 15:473–477
Ou X, Wang H, Qu D, Chen Y, Gao J, Sun H (2014) Epigenome-wide DNA methylation assay reveals placental epigenetic markers for noninvasive fetal SNP genotyping in maternal plasma. Transfusion 54:2523–2533
Ou XL, Gao J, Chen YZ, Gao Y, Qu DY, Sun HY (2013) Detecting hypermethylated fetal RASSF1A sequences in maternal plasma: implications for noninvasive paternity testing in pregnancy. Transfusion 53:1856–1858
Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR (2010) Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing. Clin Chem 56(8):1279–1286
Lo YM, Chan KC, Sun H, Chen EZ, Jiang P, Lun FM, Zheng YW, Leung TY, Lau TK, Cantor CR, Chiu RW (2010) Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med 2:61ra91
Jiang H, Xie Y, Li X, Ge H, Deng Y, Mu H, Feng X, Yin L, Du Z, Chen F, He N (2016) Noninvasive prenatal paternity testing (NIPAT) through maternal plasma DNA sequencing: a pilot study. PLoS One 11:e0159385
Zeng Z, Yan H, Wang L, Yuan E, Yang W, Liao Z, Dong Z (2010) Genome-wide screen for individual identification SNPs (IISNPs) and the confirmation of six of them in Han Chinese with pyrosequencing technology. J Forensic Sci 55:901–907
Zheng J, Xu C, Guo J, Wei Y, Ge H, Li X, Zhang C, Jiang H, Pan L, Tang W, Xie W, Zhang H, Zhao Y, Jiang F, Chen S, Wang W, Xu X, Chen F, Huang H, Jiang H (2013) Effective noninvasive zygosity determination by maternal plasma target region sequencing. PLoS One 8:e65050
Yoo J, Lee Y, Kim Y, Rha SY, Kim Y (2008) SNPAnalyzer 2.0: a web-based integrated workbench for linkage disequilibrium analysis and association analysis. BMC Bioinformatics 9:290
Liao GJ, Lun FM, Zheng YW, Chan KC, Leung TY, Lau TK, Chiu RW, Lo YM (2011) Targeted massively parallel sequencing of maternal plasma DNA permits efficient and unbiased detection of fetal alleles. Clin Chem 57:92–101
Sun YD, Cao LP (2015) Forensic validation of the goldeneye™ DNA ID 25A kit. Fa Yi Xue Za Zhi 31(4):284–286 Chinese
Guo F, Zhou Y, Song H, Zhao J, Shen H, Zhao B, Liu F, Jiang X (2016) Next generation sequencing of SNPs using the HID-ion AmpliSeq™ identity panel on the ion Torrent PGM™ platform. Forensic Sci Int Genet 25:73–84
Wienzek-Lischka S, Krautwurst A, Fröhner V, Hackstein H, Gattenlöhner S, Bräuninger A, Axt-Fliedner R, Degenhardt J, Deisting C, Santoso S, Sachs UJ, Bein G (2015) Noninvasive fetal genotyping of human platelet antigen-1a using targeted massively parallel sequencing. Transfusion 55:1538–1544
Amigo J, Salas A, Phillips C (2011) ENGINES: exploring single nucleotide variation in entire human genomes. BMC Bioinformatics 12:105
Amorim A, Pereira L (2005) Pros and cons in the use of SNPs in forensic kinship investigation: a comparative analysis with STRs. Forensic Sci Int 150:17–21
Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, Wainscoat JS, Johnson PJ, Chang AM, Hjelm NM (1998) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 62:768–775
Dhallan R, Au WC, Mattagajasingh S, Emche S, Bayliss P, Damewood M, Cronin M, Chou V, Mohr M (2004) Methods to increase the percentage of free fetal DNA recovered from the maternal circulation. JAMA 291:1114–1119
Li Y, Zimmermann B, Rusterholz C, Kang A, Holzgreve W, Hahn S (2004) Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms. Clin Chem 50:1002–1011
Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14:579–584
Tsai H, Howell T, Nitcher R, Missirian V, Watson B, Ngo KJ, Lieberman M, Fass J, Uauy C, Tran RK, Khan AA, Filkov V, Tai TH, Dubcovsky J, Comai L (2011) Discovery of rare mutations in populations: TILLING by sequencing. Plant Physiol 156:1257–1268
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of People’s Republic of China (Grants 81471825 and 81001352) and the Fundamental Research Funds for the Central Universities (12YKPY04). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors wish to thank Qing Yang and Yong Chen at Thermo Fisher Scientific for the technical supports.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
The authors declare that they have no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Human Subjects Committee at the Zhongshan School of Medicine, Sun Yat-sen University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Rights and permissions
About this article
Cite this article
Yang, D., Liang, H., Lin, S. et al. An SNP panel for the analysis of paternally inherited alleles in maternal plasma using ion Torrent PGM. Int J Legal Med 132, 343–352 (2018). https://doi.org/10.1007/s00414-017-1594-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00414-017-1594-6