Page | 1 For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. For licensing and limited use restrictions visit thermofisher.com/HIDlicensing

TECHNICAL NOTE Achieve High Resolution Y-Chromosome Haplogroup Determination with the Ion AmpliSeq™ HID Y-SNP Research Panel v1 The purpose of this technical note is to introduce HID laboratories to the new Ion AmpliSeq HID Y-SNP Research Panel v1, an MPS-based investigative panel developed in collaboration with leading European forensic genetics researchers to provide the forensic community with a high resolution Y haplogroup tool that targets 781 Y-SNPs in a single MPS assay1. This technical note informs the community of the following application of this large Y-SNP panel:

o Benefits of MPS methods for high resolution Y-SNP analysis to analyze paternal lineages and biogeographic ancestry

o Performance data for the Ion AmpliSeq HID Y-SNP Research Panel v1 to highlight the following: o Sequencing run metrics (ISP loading, total useable reads, etc.) o Marker read coverage o Concordance, sensitivity and specificity

o Workflow and system compatibility using the Ion ChefTM System and Ion GeneStudioTM S5 Systems, and Torrent Suite™ Software v5.10/v5.12

o Genotyping and Y-haplogroup analysis workflow using: o Applied Biosystems™ ConvergeTM Software v2.22 o Clean Tree Software v23

o Ordering information for the Ion AmpliSeq HID Y-SNP Research Panel v1 and other HID community panels available through Ion AmpliSeq Designer

SNP Analysis using Targeted MPS Applications With the introduction of single nucleotide polymorphism (SNP) analysis on NGS platforms, HID laboratories can now routinely ascertain biogeographic ancestry, paternal and maternal lineages4 and phenotypic5 traits using informative genetic marker sets to assist investigators with new types of forensic investigative leads. High resolution analysis of Y chromosome haplogroups using large Y-SNP panels allow improved paternal lineage identification and biogeographic ancestry inference over current commercially available panels containing smaller subsets of Y-SNPs. Using massively parallel sequencing (MPS) and Ion AmpliSeq chemistry, a single assay – capable of simultaneously interrogating hundreds of paternal lineage markers – can generate high resolution Y haplogroups in a simple workflow with challenging forensic samples due to the scalable multiplexing capacity and small amplicon panel designs. In addition to the technical advantages of MPS over single base extension (SBE) methods – which limit the number of markers that can be included in a reaction – targeted Y-SNP MPS assays offer a deeper understanding of paternal lineages to associate potentially very distant relatives in a single assay. By contrast, Y-STR methods, typically used to make paternal associations between close relatives, do not have the same resolution of large Y-SNP assays. Further, other MPS methods that focus on whole exome, whole genome or partial Y chromosome sequencing have limited value and practical use for targeted forensic applications due to the excess data obtained and DNA sample input requirements. The Ion AmpliSeq HID Y-SNP Research Panel v1 described in this technical note was designed specifically for robust analysis of challenging forensic samples (low quality and quantity) to achieve high resolution of more than 600 Y haplogroups. Developed in collaboration with European forensic genetics researchers at Erasmus

Page | 2 For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. For licensing and limited use restrictions visit thermofisher.com/HIDlicensing

University in the Netherlands, the Ion AmpliSeq HID Y-SNP Research Panel v1 provides reproducible haplotypes for 781 Y-SNPs with an average amplicon size of 130bp, using as little as 250 pg of genomic DNA input. Data presented herein are derived from in-house verification testing using an Ion Chef/S5 workflow with the final formulation of the manufactured panel lot to ensure robust performance prior to making the panel available for purchase. Refer to the Ralf et al. 2019 publication cited below for more detailed information on the Y-SNP panel development and design, assay performance, empirical test results, analysis pipeline and population sample testing. Methods For these verification studies, 12 male samples from the International Genome Sample Resource (IGSR) repository (refer to Table 1) and two control DNA samples (007 and 9948A) were used for library preparation for a total of 14 samples. Male samples from the IGSR repository were chosen to cover six population groups (Finnish in Finland, Han Chinese, Indian Telugu in the UK, Esan from Nigeria, Colombian from Medellin, Columbia, and Peruvian in Peru). To assess sensitivity of the assay, genomic DNA (gDNA) input amounts of 1 ng – 5 pg were tested using 9948 control DNA. Assay specificity was examined by female cross-reactivity testing using three female samples from the IGSR repository at 10 and 15 ng of DNA input (refer to Table 1). Female cross-reactivity of the panel was further examined with a male:female titration using 007 control DNA at 1 ng and 9947A at varying DNA amounts of 5 ng – 120 ng for library construction. Library preparation was performed with the Precision ID DL8 Kit using the Ion AmpliSeq HID Y-SNP Research Panel v1 and the Ion Chef System following the custom SNP panel protocol outlined in the Precision ID SNP Panels with the HID Ion S5/HID Ion GeneStudio S5 System Application Guide (MAN0017767). Table 1. Samples Selected from International Genome Sample Resource (IGSR)

Male Sample 1000 Genomes Population Group Reported Ancestry HG00371 Finnish in Finland European HG00372 Finnish in Finland European HG00382 Finnish in Finland European HG00421 Han Chinese East Asian HG00418 Han Chinese East Asian HG03367 Esan from Nigeria African HG03373 Esan from Nigeria African HG04017 Indian Telugu in the UK Southeast Asian HG04022 Indian Telugu in the Uk Southeast Asian HG02299 Peruvian in Peru Admixture HG02291 Peruvian in Peru Admixture HG01550 Colombian from Medellin, Colombia Admixture

Female Sample 1000 Genomes Population Group Reported Ancestry HG03369 Esan from Nigeria African HG00422 Han Chinese East Asian HG04026 Indian Telugu in the UK Southeast Asian

For library preparation, 150 uL of the Ion AmpliSeq HID Y-SNP Research Panel v1 was added to Position A and B tubes of the DL8 reagents cartridge as described in the user guide. A DNA input amount of 1 ng was used for library preparation sample set-up with the exception of the sensitivity titration study. Cycling parameters for library preparation on the Ion Chef were modified to accommodate for the number of amplicons in the primer pool (Table 2).

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Table 2. Library Preparation Parameters

Ion Chef Library Preparation Library Preparation Cycle Number 20 cycles Anneal & Extension Time 4 minutes

Manual Library Preparation Library Preparation Cycle Number 19 cycles Anneal & Extension Time 4 minutes

Library pools were quantified using the Ion Library TaqMan Quantitation Kit, diluted and pooled to 30 pM for templating on an Ion 530™ Chip using the Ion S5 Precision ID Chef & Sequencing kit. Template plans were created following the protocol outlined in the ‘Create a Planned Run for the Custom Ion AmpliSeq™ SNP Panel’ section of the Precision ID SNP Panels with the HID Ion S5/HID Ion GeneStudio S5 System Application Guide (MAN0017767) (Figure 1). Corresponding target and hotspot region BED files were used to setup templating runs (Table 3). Note that experiments performed using manual library preparation also followed the instructions provided in the Precision ID SNP Panels application guide mentioned above.

Table 3. Target & Hotspot Region BED Files Type Name

Target Region File Ion_AmpliSeq_HID_Y-SNP_Research_Panel_targets_v1.0.bed Hotspot File Ion_AmpliSeq_HID_Y-SNP_Research_Panel_hotspot_v1.0.bed

Figure 1. Templating plan set-up for the Ion AmpliSeq HID Y-SNP Research Panel v1 (custom SNP Panel). Ensure that the ‘HID Ion S5 Chef Templating 02’ templating protocol is selected in the templating plan (boxed in red).

Sequencing was performed on an Ion S5 XL System with Torrent Suite Software v5.10/v5.12. Converge Software v2.2 with NGS Data Analysis module v1.2 was used for SNP genotyping with the target and hotspot files outlined in Table 3. Aligned reads in the format of output BAM files were exported for each sample from

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the corresponding sequencing run on Torrent Suite Software and saved locally (Figure 2) for Y haplogroup analysis.

Figure 2. Output BAM files (boxed in red) for each sample on Torrent Suite Software.

Analysis of the Y-SNP sequence data was done through Erasmus University’s clean tree software v2. This analysis tool is publicly available online at https://cluster15.erasmusmc.nl/fmb/clean_tree_v2/. The software provides Y haplogroup predictions including a prediction quality score. Figure 3 outlines the laboratory workflow and steps required for the analysis of results with the Ion AmpliSeq HID Y-SNP Research Panel v1.

Figure 3. Analysis workflow for the Ion AmpliSeq HID Y-SNP Research Panel v1.

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Results Ralf et al. provide in-depth analysis and test results for an experimental test plan designed to demonstrate the range of performance of the Ion AmpliSeq HID Y-SNP Research Panel v1. The verification results provided in the following paragraphs show performance of the final build of the panel in our hands and are intended as a guideline for those who perform testing with this panel in their laboratories.∗ Assay Performance. For the baseline studies in the verification testing, the assay performed similarly to the results provided in Ralf et al. The 14 samples prepared at 1 ng of gDNA input resulted in average ISP loading of 92% and ~17.7M total useable reads. Table 4 shows the sequence run metrics for this verification data. Table 4. Sequencing run metrics for the Ion AmpliSeq HID Y-SNP Research Panel v1

Experiment # of Samples Multiplexed

ISP Loading

(%) % Useable

Reads Total Useable

Reads Median

bp Aligned

Reads (%)

Baseline Assay Performance 14 93% 51% 17,566,458 90 99% Templating Repeatability 14 90% 54% 17,957,286 88 99% Sensitivity 7 91% 52% 17,719,294 78 98% Specificity (Cross-Reactivity) 14 86% 36% 11,418,948 86 98%

Average coverage of the panel was 1378X ± 589X with >96% of the markers falling within 2SD of the mean (Table 5). Three of 781 Y-SNPs exhibited depth of coverage less then 100X with 2 markers (L408 & P44) resulting in coverages <90X. Table 5. Ion AmpliSeq HID Y-SNP Research Panel v1 Panel Performance

Metric Result Average coverage 1378X ± 589X % of markers within 1SD of the mean 65.56% % of markers within 2SD of the mean 96.03% Lower performing markers (<100X) L408, P44, & P117

Figure 4 shows average coverage per marker for the Y-SNPs in this panel. Orange lines in the figure show 2SD’s above and below the mean for the coverage results obtained in the baseline test.

∗ Note: The Ion AmpliSeq HID Y-SNP Research Panel v1 contains a total of 781 Y-SNPs within amplicon target regions. SNP coverage and genotyping accuracy were not assessed for 78 of the 859 Y-SNPs cited in Ralf et al. as this subset of SNPs lies within primer regions.

Page | 6 For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. For licensing and limited use restrictions visit thermofisher.com/HIDlicensing

Figure 4. Typical performance of the Ion AmpliSeq HID Y-SNP Research Panel v1 showing representative Y-SNPs from the panel. The assay exhibited a mean coverage of 1378X ± 589 with >96% of markers falling within 2SD of the mean. Sensitivity, Specificity and Concordance. 1 ng - 100 pg gDNA inputs provided 100% genotyping concordance in the sensitivity study. Ralf et al. reported reliable genotyping results with lower gDNA inputs (e.g., 100-250 pg) without a negative impact on haplogroup analysis. With the large number of amplicons in this multiplex, one would expect a decline with lower DNA input amounts; however, the panel provided full representation of SNP genotypes at 100 pg of gDNA. This result can be attributed to the higher number of reads per sample observed with the Ion S5 system. Correct Y-haplogrouping was obtained with as little as 10 pg of gDNA input despite a relatively high amount of Y-SNP marker dropout (~42%) (Table 6). One should note that dropout of SNPs may decrease the resolution of lineage analysis, but the results should continue to follow the respective branch of the Y tree. Table 6. Sensitivity testing of the Ion AmpliSeq HID Y-SNP Research Panel v1 using 007 control DNA

gDNA input (pg)

Aligned reads

Called Y-SNPs

Number of Dropout Y-

SNPs

Reported Haplogroup

Most downstream

Y-SNP Marker 1000 3,791,288 781 0 R1b1a1a2a1a2c1a Z2542 750 2,930,758 781 0 R1b1a1a2a1a2c1a Z2542 500 3,362,110 781 0 R1b1a1a2a1a2c1a Z2542 250 3,049,854 781 0 R1b1a1a2a1a2c1a Z2542 100 2,320,937 781 0 R1b1a1a2a1a2c1a Z2542 10 744,076 448 333 R1b1a1a2a1a2c1a Z2542 5 577,789 226 555 R1b1a1a2a1a2c1 L21

Y-SNP markers demonstrating lower relative coverage (<300X coverage) across the 14 population samples tested were identified and shown in Table 7. These lower performing Y-SNP markers are at higher risk of dropping out as the number of samples per 530 chip are increased above 14 samples. Ralf et al. note that Y-SNP markers with <20X coverage are likely to show the correct genotype due to their haploid nature and the

0

500

1000

1500

2000

2500

3000

3500

4000

4500L4

08Z2

356

Y192

32 M2

M18

4L1

034

Z222

0Z1

40CT

S616

Z204

1U

198

CTS5

95L9

02S2

3458

L554

Z220 L5

3P3

05U

175

M40

M23

7L5

27F6

50F5

73AM

0090

3Z5

2D

F23

L90

P197

M48

5Z4

452

M42

3Z7

8L1

089

F120

5Z1

46L2

75JS

T022

457

L708

Z283

Z599

4L9

81CT

S446

6CT

S480

3L5

69 Z63

Z420

28M

75 L68

Page

23Y1

8596

Z296

1F3

243

PF33

59Y1

336

V65

L314

Page

116

M11

6Z2

88M

12Z6

47P1

20L1

120

P15

Z243

2F1

876

V69

Z93

Z445

12U

250

CTS4

90M

293

L232

L260

V171 V7

8Z9

469

M83

48FG

C330

62M

178

L723

M21

4Z7

700

L130

7M

180

P405

Z592

6M

109

M69

M46

M55

M29

40M

258

P92

P262

CTS6

364

M20

1

Aver

age

Read

Dep

th (n

=14)

ySNP

Ion AmpliSeq HID YSNP Research Panel v1 - Average Marker Coverage

Marker Coverage Average Marker Cov Upper 2 SD Lower 2 SD

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absence of heterozygotes in single source samples. Laboratories are encouraged to perform internal testing to identify the minimum marker coverage thresholds to ensure accurate genotyping and Y-haplogroup analysis for sample types analyzed with the Ion AmpliSeq HID Y-SNP Research Panel v1. Table 7. Y-SNP markers with low overall coverage (<300X)

Y-SNP Average Coverage (n=14)

L408 64 P44 72 P117 94

M104_1 108 PF5172 198

Haplogroup and haplotype concordance for control DNA 007 and 9948A were compared to results obtained by Ralf et al 2019 (Tables 8 and 9). Likewise, the IGSR sample set was also examined for haplogroup and haplotype concordance. In both instances, 100% concordance was obtained for these control sample sets thereby demonstrating accuracy and inter-laboratory assay repeatability. Table 8. Concordance results for Thermo Fisher verification testing of AmpliSeq HID Y-SNP Research Panel v1 and Ralf et al. 2019 publication

Sample Reported

Haplogroup (TFS)

Most Downstream Marker (TFS)

Reported Haplogroup

(Ralf et al 2019)

Most Downstream Marker

(Ralf et al 2019) 007 R1b1a1a2a1a2c1a Z2542 R1b1a1a2a1a2c1a Z2542

9948A R1b1a1a2a1a1c1 Z156 R1b1a1a2a1a1c1 Z156 Table 9. Concordance results for the 14 samples tested (IGSR and control samples)

Sample 1000 Genomes Population Group Haplogroup Most Downstream Marker

007 - R1b1a1a2a1a2c1a Z2542 9948A - R1b1a1a2a1a1c1 Z156

HG00371 Finnish in Finland N1a1a1a1a2a1a Z1935 HG00372 Finnish in Finland I1a1b1a4a Z74 HG00382 Finnish in Finland R1a1a1b1a2a Z92 HG00421 Han Chinese O2a1c1a1a1a F11 HG00418 Han Chinese O1b1a1a M95 HG04017 Indian Telugu in the UK R1a1a1b2a1a1 Y8 HG04022 Indian Telugu in the UK R2a2b1b2b3b2a1 FGC18152 HG03367 Esan from Nigeria E1b1a1a1a1c1a1a U174 HG03373 Esan from Nigeria E1b1a1a1a2a1a3b1a1 U181 HG02299 Peruvian in Peru Q1b1a1a CTS11970 HG02291 Peruvian in Peru Q1b1a1a CTS11970 HG01550 Colombian from Medellin, Colombia R1b1a1a2a1a1c2b2b1a Z326

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Assay specificity was examined utilizing female IGSR samples with a gDNA Input of 15 ng and 10 ng. A low percentage of Y-SNPs (0.26% - 1.41%) was called in the female samples tested (HG03369, HG04026, and HG04022) (data not shown). This result may be attributed to sequence homology between the X and Y chromosomes (as reported in Ralf et al.). Similarly, a male:female titration study was performed to determine the ability to accurately call a Y-haplogroup from a male donor in the presence of excess female gDNA (Table 10). Results from the male:female titration study demonstrated the ability to correctly detect the male donor haplogroup in the presence of up to a 120-fold excess of female DNA. Despite the high percent of accurately genotyped Y-SNPs, the observed Y-SNP coverage overall was low in this male:female titration series. Table 10. Reported haplogroups from clean tree v2 for the male:female sensitivity titration results

Similar genotyping results were obtained through Converge v2.2 and clean tree with the exception of No Calls (N) which occurred when a SNP did not meet Converge quality thresholds (e.g. low coverage or poor quality reads).

Figure 5. Sample genotyping results for the Ion AmpliSeq HID Y-SNP Research Panel v1 on Converge v2.2.

007 DNA Amount

9947A DNA Amount Total Reads

Called Y-SNPs (% of Total

SNPs) Average

Coverage Reported Haplogroup

from Clean Tree v2

1 ng

5 ng 844,422 767 (98.2%) 161 ± 78 R1b1a1a2a1a2c1a 10 ng 644,605 740 (94.7%) 69 ± 33 R1b1a1a2a1a2c1a 20 ng 580,681 596 (76.3%) 37 ± 20 R1b1a1a2a1a2c1 40 ng 526,894 602 (77.1%) 34 ± 18 R1b1a1a2a1a2c1a 80 ng 554,736 428 (54.8%) 24 ± 17 R1b1a1a2a1a2c1

120 ng 601,953 309 (39.6%) 20 ± 20 R1b1a1a2a1a2c1

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Figure 6. Haplogroup prediction output in tab-delimited format from clean tree v2. The tab-delimited output file displays sample haplogroup, most downstream marker, and QC scores.

Ordering information. To obtain the Ion AmpliSeq HID Y-SNP Research Panel v1, customers should visit Ion AmpliSeq Designer on thermofisher.com to create a user account, login and purchase the panel through the website. The panel is catalogued under the Community Panel section of Ion AmpliSeq Designer.

Figure 7. Ion AmpliSeq HID Y-SNP Research Panel v1 is available on Ion AmpliSeq Designer. The direct link to the Y-SNP Community Panel ordering page can be found here.

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Table 11. Ordering information for Ion AmpliSeq HID Y-SNP Research Panel v1

Number of Primer Pairs per Pool

Primer Concentration Minimum Pack Size Quantity per Box

96 < x ≤ 3072 100 nM each 3,000 rxns (20 tubes @ 150 rxns/tube)

Up to 80 tubes (12,000 rxns total)

Conclusions Iterative panel development efforts between Thermo Fisher Scientific and Erasmus University have created a robust and informative Ion AmpliSeq HID Y-SNP Research Panel v1 that contains 602 amplicons and a total of 781 Y-SNP targets. The selected Y-SNPs in this assay show practical utility in forensic investigations given the high resolution lineage analysis and small amplicon design as described in Ralf et al. The panel performance data provided in this technical note provides a guideline for researchers and academicians interested in testing the panel with population and forensic samples. For this study, 14 samples per Ion 530 Chip provided suitable results with a high percentage (>96%) of Y-SNPs exhibiting >60X coverage. This coverage threshold was noted to identify lower performing Y-SNPs in the panel and was not intended as a value to determine signal versus noise. Given the haplotypic nature of this panel, one could reasonably establish a lower analytical threshold with minimal risk of obtaining incorrect DNA types. However, consideration should be given when decreasing genomic DNA input amounts <1 ng or increasing sample throughputs as such modifications may lead to increased marker dropout. In this regard, Ralf et al. show a drop in panel performance in the 100 - 250 pg range. However, only a loss of resolution for the haplogroup assignment was observed even at the 5 pg gDNA input level, which exhibited a dropout of ~44% of the markers in the panel. Therefore, dropout of a moderate percentage of Y-SNP markers will likely follow the correct branches of the Y tree and provide accurate Y haplogroup results due to the total Y-SNP number and overlapping SNPs in the panel. Users are advised to perform empirical studies to support protocol modifications and assess the inherent variability of the workflow which may further impact results obtained. One obvious benefit of targeting Y chromosome SNPs is the ability to obtain genotype information from a male contributor without interference from female DNA. In these male:female mixture scenarios, ideally non-specific amplification signal from the female DNA does not impact the male Y-SNP results. This attribute is especially important when applying the assay in casework examinations such as sexual assaults when male:female mixtures are likely to arise. However, given that this assay contains a high level of multiplexing (602 total target amplicons), the small amount of nonspecific amplification detected in our study was not unexpected due to sequence homology between the X and Y chromosomes. The verification results presented in this study demonstrate baseline assay performance and interlaboratory concordance results with Forensic Y-SNP analysis beyond SNaPshot 2019 publication by Ralf et al. for the final build of Ion AmpliSeq HID Y-SNP Research Panel v1 formulation available on Ion AmpliSeq Designer. The full panel development results published in the Ralf et al. publication show a broader range of experimental conditions, including results from degraded DNA, direct amplification, mixture analysis, and population sample testing. In order to move the Y-SNP Research Panel v1 into a caseworking context, underlying population reference data are needed to demonstrate the utility of the panel, standardize marker selection and refine global comparisons between population haplogroups. The high resolution panel described in this note offers a standardized reference potential from human populations around the world to increase utility of ancestry inferences as well as paternal lineage identification.

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References

1 Ralf, A., Oven, M., Gonzalez, O., de Kniff, P., der Beek, K., Wootton, S., Lagace, R., Kayser, M. Forensic Y-SNP analysis beyond SNaPshot: High-resolution Y-chromosomal haplogrouping from low quality and quantity DNA using Ion AmpliSeq and targeted massively parallel sequencing. Forensic Sci Int Genet. Volume 41, July 2019, Pages 93-106. 2 Converge Software v2.2. (https://www.thermofisher.com/order/catalog/product/A35131#/A35131) 3 Clean tree software v2. (https://cluster15.erasmusmc.nl/fmb/clean_tree_v2/) 4 A. Ralf, et al., Simultaneous analysis of hundreds of Y‐Chromosomal SNP s for high‐resolution paternal lineage classification using targeted semiconductor sequencing, Hum. Mutat. 36 (1) (2015) 151–159. 5 Walsh S1, Liu F, Wollstein A, Kovatsi L, Ralf A, Kosiniak-Kamysz A, Branicki W, Kayser M. The HIrisPlex system for simultaneous prediction of hair and eye colour from DNA. Forensic Sci Int Genet. 2013 Jan;7(1):98-115. doi: 10.1016/j.fsigen.2012.07.005. Epub 2012 Aug 20.