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Non-invasive prenatal paternity testing with STRs: A pilot study
G Model FSIGSS 1112 No. of Pages 2
Forensic Science International: Genetics Supplement Series xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Non-invasive prenatal paternity testing with STRs: A pilot study M. Gysi* , N. Arora, A. Sulzer, P. Voegeli, A. Kratzer Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 September 2015 Accepted 18 September 2015 Available online xxx
Non-invasive prenatal paternity testing using foetal cell-free DNA (cfDNA) in the mother's blood eliminates the risks of miscarriage associated with invasive methods such as chorionic villus sampling and amniocentesis. However, the low abundance of cfDNA as well as the large excess of maternal cfDNA makes foetal DNA typing very challenging. As illustrated by our case study, SNP typing at 317,000 positions using cfDNA in the mother’s blood was shown to successfully identify the father. However, for forensic routine work, STR analysis would be preferable over SNP analysis, as for STR markers the frequencies and population data are available and the statistics are well established. We therefore initiated a pilot study to set up a next-generation sequencing (NGS) assay using an early access version of the Ion AmpliSeq HID STR 25-plex on the Ion PGMTM. We successfully confirmed paternity from blood samples from two pregnant women with 10 and 16 weeks of gestation (GW). Drop-outs were not observed but further testing is necessary for a full evaluation. Overall, our data suggest that paternity testing with STRs and NGS is a promising approach for non-invasive prenatal paternity testing. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Non-invasive prenatal paternity STR NGS Ion PGM
1. Introduction Current non-invasive prenatal paternity tests rely on SNP [1] and Y-STR typing [2]. Attempts to type foetal autosomal STRs in the mother’s blood have so far not been successful [2,3]. The large excess of maternal cfDNA – up to 50 times more [4] – as well as the highly fragmented cfDNA – most of the fragments are smaller than 250 base pairs – are the main obstacles to typing foetal autosomal STRs. In one of our forensic cases, the prosecution requested noninvasive prenatal paternity testing to investigate potential sexual misconduct by a prison guard. He had committed suicide, allegedly after leaving a woman under his supervision pregnant. As the woman refused invasive sampling, foetal cfDNA from maternal blood was analysed by sequencing 317,000 SNPs on an Illumina Infinium microarray assay at Natera, inc. (USA). Natera used their bioinformatics tool “Parental Support” to calculate a paternity probability of >99.9% for the alleged father. Paternity was confirmed in our lab with postnatal STR profiling, with a probability of >99.9999%, agreeing with Natera’s SNP results. Although SNP analysis was successfully used to identify the father, it remains unclear whether this analysis can be offered routinely in forensics, as some SNPs might lie in coding regions and/or may be associated with diseases.
* Corresponding author. E-mail address: [email protected] (M. Gysi).
With the increasing availability of commercial NGS applications for STR analysis, the new technology has the potential to circumvent the challenges of fragmented DNA and low mixture ratios for three reasons: (1) with NGS, size separation of STR-loci is no longer required thus enabling the use of mini STR markers, (2) mixtures can be deconvoluted to ratios smaller than 1:20 [5] and (3) repeat motif variations allow distinction of foetal peaks masked by peaks or stutters from the maternal profile [5]. Our first findings with an early access version of the Ion AmpliSeq HID STR 25-plex (Applied Biosystems1, AB) on the Ion PGMTM (AB) [6] mostly confirm these advantages. 2. Materials and methods Five ml EDTA blood samples from two pregnant women at 10 and 16 weeks of gestation (GW) were taken with written consent. After plasma separation (10 min 2000g followed by 3 min 15,500g), cfDNA was extracted from blood plasma using the QiaAmp MinElute Virus Spin Kit (Qiagen) following the manufacturer’s instructions with one modification: we incorporated 200 ml blood plasma into the column twice for a total of 400 ml. Samples were eluted in 25 ml resulting in 44 and 91.8 pg/ml cfDNA for mother 1 and 2, respectively. Ten microliters were used for STR amplification with both GlobalFilerTM (AB) [7] and Ion AmpliSeq HID STR 25-plex (AB) performing 30 PCR cycles for each. For reference samples (buccal swabs from the fathers and blood pellet fractions from the mothers), 1 ng DNA was used for PCR. Capillary electrophoresis (CE) analysis and Ion AmpliSeq library
http://dx.doi.org/10.1016/j.fsigss.2015.09.115 1875-1768/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: M. Gysi, et al., Non-invasive prenatal paternity testing with STRs: A pilot study, Forensic Sci. Int. Gene. Suppl. (2015), http://dx.doi.org/10.1016/j.fsigss.2015.09.115
G Model FSIGSS 1112 No. of Pages 2
e2
M. Gysi et al. / Forensic Science International: Genetics Supplement Series xxx (2015) xxx–xxx
Table 1 Comparison of a CE analysis (GlobalFilerTM) and an NGS analysis (Ion AmpliSeq HID STR 25-plex on the PGMTM) of cfDNA from two pregnant women’s blood plasma samples. CE (GlobalFilerTM)
NGS (early access Ion AmpliSeq HID STR 25-plex)
Distinct paternal Masked by maternal alleles allele/stutter Mother 1 4 (16 GW) Mother 2 (10 4 GW)
Dropouts
Dropins
Distinct paternal alleles
Masked by maternal allele/stutter
Dropouts
Dropins
Repeat motif variants (of masked alleles)
11
6
0
9
15
0
0
2
13
4
0
12
12
0
0
0
preparations were performed according to the manufacturer’s recommendations. A total of 315,000 and 460,000 reads were obtained for the two plasma samples and 15,000 and 18,000 reads for the two reference samples on the Ion PGMTM. Data were analysed with the HID-STR Genotyper Plugin v3.0 (AB). The default value of 30 reads was used as a threshold for allele calling, offladder alleles of 0.1 and 0.3 were treated as sequencing or data analysis errors rather than true alleles.
The probabilities would increase if markers with masked paternal alleles were included into calculations. Masked paternal alleles with repeat motif variants (2 of the 15 masked alleles in sample 1) were not taken into account as frequency data for repeat motif variants have yet to be determined. Paternal alleles in stutter positions were also excluded from the calculation, regardless of the stutter peak height, as stutter ratios were not available yet. 4. Conclusions
3. Results and discussion In our pilot study, blood plasma samples from two pregnant women were analysed with an early access version of the Ion AmpliSeq HID STR 25-plex on the Ion PGMTM to screen for foetal cfDNA. The results were compared to CE analysis using GlobalFilerTM. A cfDNA profile from a pregnant woman’s blood has to be interpreted as a mother-child mixture with the mother being the major contributor, in 20–50 fold excess. For every marker, the maternal allele of the foetus is therefore masked by the mother’s profile, while the paternal allele is expected to be visible as the minor contributor when not masked by a mother’s allele or stutter. The highly fragmented cfDNA is represented by a CE DNA profile typical of degraded DNA. Therefore only the smallest markers of every dye display the foetal alleles. Nonetheless, for both of the blood samples we examined, we were able to detect at least 4 distinct paternal alleles at 21 markers with the GlobalFilerTM Kit (Table 1). This result contrasts with that in previous studies, where only amelogenin was successfully typed for the foetus in an autosomal STR analysis [2]. It must be noted, however, that at least 4–6 drop-outs for the foetus from mother 2 and mother 1, respectively, were observed. This number is probably an underestimate, as in cases where the mother and the father share at least one allele it is unclear whether the paternal allele is masked by the maternal allele or whether it dropped out. Compared to CE, the advantages of typing mini-STRs with NGS are evident, as we detect a higher number of foetal alleles. Moreover, we could not confirm any drop-outs in this study (Table 1). Nonetheless, our results are still preliminary: a more accurate assessment of drop outs requires a comparison with the true foetal genotypes following post-natal STR typing. For each foetus, we estimated paternity probabilities using only the set of markers with distinct foetal alleles. We took all possible foetal genotype combination for these markers into account: Mother 1 and Father 1: probability of paternity > 99.9997% (number of markers = 9) Mother 2 and Father 2: probability of paternity > 99.9999997% (number of markers = 12)
In order to increase accuracy, future large-scale validation studies will have to show that drop-out events occur very rarely. Moreover, inclusion of drop-out probabilities in the statistics may need to be considered as proposed by Balding and Buckleton [8] to account for the low template nature of cfDNA. Stutter ratios as well as signal noise have to be evaluated for each STR marker separately to eventually come up with a set of robust markers. Overall, the high number of detected paternal alleles as well as the low number of drop-outs obtained in our pilot study highlight the potential of this novel technique in forensic paternity testing. Funding None. Conflict of interest None. References [1] A. Ryan, et al., Informatics-based, highly accurate, noninvasive prenatal paternity testing, Genet. Med. 15 (6) (2013) 473–477. [2] J. Wagner, et al., Non-invasive prenatal paternity testing from maternal blood, Int. J. Legal Med. 123 (1) (2009) 75–79. [3] L. Birch, et al., Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation, Clin. Chem. 51 (2) (2005) 312–320. [4] H.C. Fan, et al., Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing, Clin. Chem. 56 (8) (2010) 1279–1286. [5] S.L. Fordyce, et al., Second-generation sequencing of forensic STRs using the Ion Torrent HID STR 10-plex and the Ion PGM, Forensic Sci. Int. Genet. 14 (2015) 132–140. [6] J.M. Rothberg, et al., An integrated semiconductor device enabling non-optical genome sequencing, Nature 475 (7356) (2011) 348–352. [7] D.Y. Wang, et al., Developmental validation of the GlobalFiler Express PCR Amplification Kit: A 6-dye multiplex assay for the direct amplification of reference samples, Forensic Sci. Int. Genet. 19 (2015) 148–155. [8] D.J. Balding, J. Buckleton, Interpreting low template DNA profiles, Forensic Sci. Int. Genet. 4 (1) (2009) 1–10.
Please cite this article in press as: M. Gysi, et al., Non-invasive prenatal paternity testing with STRs: A pilot study, Forensic Sci. Int. Gene. Suppl. (2015), http://dx.doi.org/10.1016/j.fsigss.2015.09.115
Forensic Science International: Genetics Supplement Series xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Non-invasive prenatal paternity testing with STRs: A pilot study M. Gysi* , N. Arora, A. Sulzer, P. Voegeli, A. Kratzer Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 September 2015 Accepted 18 September 2015 Available online xxx
Non-invasive prenatal paternity testing using foetal cell-free DNA (cfDNA) in the mother's blood eliminates the risks of miscarriage associated with invasive methods such as chorionic villus sampling and amniocentesis. However, the low abundance of cfDNA as well as the large excess of maternal cfDNA makes foetal DNA typing very challenging. As illustrated by our case study, SNP typing at 317,000 positions using cfDNA in the mother’s blood was shown to successfully identify the father. However, for forensic routine work, STR analysis would be preferable over SNP analysis, as for STR markers the frequencies and population data are available and the statistics are well established. We therefore initiated a pilot study to set up a next-generation sequencing (NGS) assay using an early access version of the Ion AmpliSeq HID STR 25-plex on the Ion PGMTM. We successfully confirmed paternity from blood samples from two pregnant women with 10 and 16 weeks of gestation (GW). Drop-outs were not observed but further testing is necessary for a full evaluation. Overall, our data suggest that paternity testing with STRs and NGS is a promising approach for non-invasive prenatal paternity testing. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Non-invasive prenatal paternity STR NGS Ion PGM
1. Introduction Current non-invasive prenatal paternity tests rely on SNP [1] and Y-STR typing [2]. Attempts to type foetal autosomal STRs in the mother’s blood have so far not been successful [2,3]. The large excess of maternal cfDNA – up to 50 times more [4] – as well as the highly fragmented cfDNA – most of the fragments are smaller than 250 base pairs – are the main obstacles to typing foetal autosomal STRs. In one of our forensic cases, the prosecution requested noninvasive prenatal paternity testing to investigate potential sexual misconduct by a prison guard. He had committed suicide, allegedly after leaving a woman under his supervision pregnant. As the woman refused invasive sampling, foetal cfDNA from maternal blood was analysed by sequencing 317,000 SNPs on an Illumina Infinium microarray assay at Natera, inc. (USA). Natera used their bioinformatics tool “Parental Support” to calculate a paternity probability of >99.9% for the alleged father. Paternity was confirmed in our lab with postnatal STR profiling, with a probability of >99.9999%, agreeing with Natera’s SNP results. Although SNP analysis was successfully used to identify the father, it remains unclear whether this analysis can be offered routinely in forensics, as some SNPs might lie in coding regions and/or may be associated with diseases.
* Corresponding author. E-mail address: [email protected] (M. Gysi).
With the increasing availability of commercial NGS applications for STR analysis, the new technology has the potential to circumvent the challenges of fragmented DNA and low mixture ratios for three reasons: (1) with NGS, size separation of STR-loci is no longer required thus enabling the use of mini STR markers, (2) mixtures can be deconvoluted to ratios smaller than 1:20 [5] and (3) repeat motif variations allow distinction of foetal peaks masked by peaks or stutters from the maternal profile [5]. Our first findings with an early access version of the Ion AmpliSeq HID STR 25-plex (Applied Biosystems1, AB) on the Ion PGMTM (AB) [6] mostly confirm these advantages. 2. Materials and methods Five ml EDTA blood samples from two pregnant women at 10 and 16 weeks of gestation (GW) were taken with written consent. After plasma separation (10 min 2000g followed by 3 min 15,500g), cfDNA was extracted from blood plasma using the QiaAmp MinElute Virus Spin Kit (Qiagen) following the manufacturer’s instructions with one modification: we incorporated 200 ml blood plasma into the column twice for a total of 400 ml. Samples were eluted in 25 ml resulting in 44 and 91.8 pg/ml cfDNA for mother 1 and 2, respectively. Ten microliters were used for STR amplification with both GlobalFilerTM (AB) [7] and Ion AmpliSeq HID STR 25-plex (AB) performing 30 PCR cycles for each. For reference samples (buccal swabs from the fathers and blood pellet fractions from the mothers), 1 ng DNA was used for PCR. Capillary electrophoresis (CE) analysis and Ion AmpliSeq library
http://dx.doi.org/10.1016/j.fsigss.2015.09.115 1875-1768/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: M. Gysi, et al., Non-invasive prenatal paternity testing with STRs: A pilot study, Forensic Sci. Int. Gene. Suppl. (2015), http://dx.doi.org/10.1016/j.fsigss.2015.09.115
G Model FSIGSS 1112 No. of Pages 2
e2
M. Gysi et al. / Forensic Science International: Genetics Supplement Series xxx (2015) xxx–xxx
Table 1 Comparison of a CE analysis (GlobalFilerTM) and an NGS analysis (Ion AmpliSeq HID STR 25-plex on the PGMTM) of cfDNA from two pregnant women’s blood plasma samples. CE (GlobalFilerTM)
NGS (early access Ion AmpliSeq HID STR 25-plex)
Distinct paternal Masked by maternal alleles allele/stutter Mother 1 4 (16 GW) Mother 2 (10 4 GW)
Dropouts
Dropins
Distinct paternal alleles
Masked by maternal allele/stutter
Dropouts
Dropins
Repeat motif variants (of masked alleles)
11
6
0
9
15
0
0
2
13
4
0
12
12
0
0
0
preparations were performed according to the manufacturer’s recommendations. A total of 315,000 and 460,000 reads were obtained for the two plasma samples and 15,000 and 18,000 reads for the two reference samples on the Ion PGMTM. Data were analysed with the HID-STR Genotyper Plugin v3.0 (AB). The default value of 30 reads was used as a threshold for allele calling, offladder alleles of 0.1 and 0.3 were treated as sequencing or data analysis errors rather than true alleles.
The probabilities would increase if markers with masked paternal alleles were included into calculations. Masked paternal alleles with repeat motif variants (2 of the 15 masked alleles in sample 1) were not taken into account as frequency data for repeat motif variants have yet to be determined. Paternal alleles in stutter positions were also excluded from the calculation, regardless of the stutter peak height, as stutter ratios were not available yet. 4. Conclusions
3. Results and discussion In our pilot study, blood plasma samples from two pregnant women were analysed with an early access version of the Ion AmpliSeq HID STR 25-plex on the Ion PGMTM to screen for foetal cfDNA. The results were compared to CE analysis using GlobalFilerTM. A cfDNA profile from a pregnant woman’s blood has to be interpreted as a mother-child mixture with the mother being the major contributor, in 20–50 fold excess. For every marker, the maternal allele of the foetus is therefore masked by the mother’s profile, while the paternal allele is expected to be visible as the minor contributor when not masked by a mother’s allele or stutter. The highly fragmented cfDNA is represented by a CE DNA profile typical of degraded DNA. Therefore only the smallest markers of every dye display the foetal alleles. Nonetheless, for both of the blood samples we examined, we were able to detect at least 4 distinct paternal alleles at 21 markers with the GlobalFilerTM Kit (Table 1). This result contrasts with that in previous studies, where only amelogenin was successfully typed for the foetus in an autosomal STR analysis [2]. It must be noted, however, that at least 4–6 drop-outs for the foetus from mother 2 and mother 1, respectively, were observed. This number is probably an underestimate, as in cases where the mother and the father share at least one allele it is unclear whether the paternal allele is masked by the maternal allele or whether it dropped out. Compared to CE, the advantages of typing mini-STRs with NGS are evident, as we detect a higher number of foetal alleles. Moreover, we could not confirm any drop-outs in this study (Table 1). Nonetheless, our results are still preliminary: a more accurate assessment of drop outs requires a comparison with the true foetal genotypes following post-natal STR typing. For each foetus, we estimated paternity probabilities using only the set of markers with distinct foetal alleles. We took all possible foetal genotype combination for these markers into account: Mother 1 and Father 1: probability of paternity > 99.9997% (number of markers = 9) Mother 2 and Father 2: probability of paternity > 99.9999997% (number of markers = 12)
In order to increase accuracy, future large-scale validation studies will have to show that drop-out events occur very rarely. Moreover, inclusion of drop-out probabilities in the statistics may need to be considered as proposed by Balding and Buckleton [8] to account for the low template nature of cfDNA. Stutter ratios as well as signal noise have to be evaluated for each STR marker separately to eventually come up with a set of robust markers. Overall, the high number of detected paternal alleles as well as the low number of drop-outs obtained in our pilot study highlight the potential of this novel technique in forensic paternity testing. Funding None. Conflict of interest None. References [1] A. Ryan, et al., Informatics-based, highly accurate, noninvasive prenatal paternity testing, Genet. Med. 15 (6) (2013) 473–477. [2] J. Wagner, et al., Non-invasive prenatal paternity testing from maternal blood, Int. J. Legal Med. 123 (1) (2009) 75–79. [3] L. Birch, et al., Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation, Clin. Chem. 51 (2) (2005) 312–320. [4] H.C. Fan, et al., Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing, Clin. Chem. 56 (8) (2010) 1279–1286. [5] S.L. Fordyce, et al., Second-generation sequencing of forensic STRs using the Ion Torrent HID STR 10-plex and the Ion PGM, Forensic Sci. Int. Genet. 14 (2015) 132–140. [6] J.M. Rothberg, et al., An integrated semiconductor device enabling non-optical genome sequencing, Nature 475 (7356) (2011) 348–352. [7] D.Y. Wang, et al., Developmental validation of the GlobalFiler Express PCR Amplification Kit: A 6-dye multiplex assay for the direct amplification of reference samples, Forensic Sci. Int. Genet. 19 (2015) 148–155. [8] D.J. Balding, J. Buckleton, Interpreting low template DNA profiles, Forensic Sci. Int. Genet. 4 (1) (2009) 1–10.
Please cite this article in press as: M. Gysi, et al., Non-invasive prenatal paternity testing with STRs: A pilot study, Forensic Sci. Int. Gene. Suppl. (2015), http://dx.doi.org/10.1016/j.fsigss.2015.09.115