Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing.

OBJECTIVES: To assess the efficacy of Y-chromosome mini-STR-based next-generation sequencing (NGS) for non-invasive prenatal paternity testing (NIPPT).
METHODS: DNA was extracted from the plasma of 24 pregnant women, and cell-free fetal DNA (cffDNA) haplotyping was performed at 12 Y-chromosome mini-STR loci using the Illumina NextSeq 500 system. The cffDNA haplotype was validated by the paternal haplotype. Subsequentlly, the paternity testing parameters were attributed to each case quantitatively.
RESULTS: The biological relationship between the alleged fathers and infants in all 24 family cases were confirmed by capillary electrophoresis (CE). The Y-chromosome mini-STR haplotypes of all 14 male cffDNA were obtained by NGS without any missing loci. The alleles of cffDNA and paternal genomic DNA were matched in 13 cases, and a mismatched allele was detected at the DYS393 locus in one case and considered as mutation. No allele was detected in the 10 female cffDNA. The combined paternity index (CPI) and probability of paternity calculation was based on 6 loci Y-haplotype distributions of a local population. The probability of paternity was 98.2699-99.8828% for the cases without mutation, and 14.8719% for the case harboring mutation.
CONCLUSIONS: Our proof-of-concept study demonstrated that Y-chromosome mini-STR can be used for NGS-based NIPPT with high accuracy in real cases, and is a promising tool for familial searching, paternity exclusion and sex selection in forensic and medical applications.

Introduction

The conventional prenatal paternity testing methods rely on invasive amniocentesis or chorionic villus sampling, which may lead to pregnancy-related complications and increase the procedure-related risk of miscarriage to 0.35% [1, 2]. Lo et al. [3] first isolated cell-free fetal DNA (cffDNA) from maternal plasma in the 1990s, which paved the way for developing non-invasive prenatal testing techniques [4]. Currently, non-invasive prenatal testing is widely used for detecting the rhesus D blood type [5], fetal aneuploidy like Down syndrome [6], fetal sex [7] and paternity [8]. Approximately 99% of the cffDNA is shorter than 313 base pairs (bp), and is largely derived from trophoblast destruction, or the apoptosis of fetal hematopoietic cells and trans-placental transfer to a lesser extent [9, 10]. A previous study showed that the circulating cffDNA had a mean half-life of 16.3 min and was undetectable in the maternal plasma 2 h post-delivery. This indicates that cffDNA testing is not affected by carryover from previous pregnancies [11].

In forensic science, Y-STR typing is used as an additional tool for confirming the paternity or identifying individual male sequences in complex DNA mixtures along with autosomal STR typing [12, 13]. Due to its uniparental inheritance, the results of Y-STR typing are interpreted on the basis of haplotype distribution, which reveals the familial relationship pattern [14]. A major challenge in non-invasive prenatal paternity testing (NIPPT) is the detection of fetal-specific markers from the paternal genetic material in the maternal plasma. Therefore, autosomal STR-based prenatal paternity testing is not used frequently due to maternal DNA contamination and the requirement of short target sequences. Since Y-specific cffDNA can be easily distinguished from the abundant maternal DNA signals in the maternal plasma, detection of Y-chromosome markers can be used to assess the paternity of male fetuses by capillary electrophoresis (CE) or SNP-based next-generation sequencing (NGS) [15, 16]. In addition, mini-STR loci detection is an effective strategy for recovering genetic information from highly degraded DNA samples, and was solely used to fingerprint 20% of the degraded DNA samples from the aftermath of the 9/11 World Trade Centre terrorist attacks with reduced PCR amplicon sizes [17, 18]. The aim of our study was to evaluate the efficacy of Y-chromosome mini-STR-based NGS for NIPPT, and quantify the extent of match using paternity testing parameters.

Materials and methods

Sample collection

Peripheral blood samples were collected from 24 pregnant women undergoing prenatal tests, and their male partners, at the Dalian Blood Centre from April 2018 to December 2019. The age of the women ranged from 26–38 years, and the gestational age ranged from 21–37 weeks (17 women in the second trimester and 7 in the third trimester). Buccal swabs were collected from the infants after delivery. All participants signed the informed consent, and the study was approved by the Dalian Blood Centre Ethics Committee.

DNA extraction

Around 5 mL peripheral blood was collected from the pregnant women in anticoagulant-treated tubes. Plasma was isolated from the whole blood sample using two-step centrifugation at 1500 ×g for 10 min and 13,000 ×g for 10 min [19]. The supernatant was collected and stored at -80°C. Plasma DNA was extracted from 2 mL cell-free maternal plasma using the MagPure Circulating DNA Maxi Kit (Angen Biotech, Guangzhou, China) and eluted with 60 μL water according to the manufacturer’s instructions. The 2800M Control DNA (Promega, Madison, WI, USA) was used as the positive control, and nuclease-free water was used as the PCR blank. Genomic DNA was extracted from the peripheral blood of the pregnant women and their husbands, and from the buccal swabs of infants using HiPure Tissue & Blood DNA Kit (Angen Biotech). The quantity and purity of the 1 μL extracted DNA were determined by sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with Tanon 1600 Gel Imaging System (Shanghai Tanon Science & Technology, Shanghai, China) and NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA) respectively. Presence of contaminants such as RNA and proteins can increase the absorbance of the DNA samples at 260, resulting in overestimation of DNA concentration. Therefore, spectrophotometric measurement of DNA was supplemented with SDS-PAGE to avoid interference from these contaminants. The DNA concentration of each sample was estimated on the basis of a molecular weight marker band.

Paternity testing by Capillary Electrophoresis (CE)

Paternity testing was performed using the Microreader™ 21 ID System (Microread Genetics, Beijing, China) and Microreader™ 29Y ID System (Microread Genetics, Beijing, China). The genomic DNA extracted from all subjects was amplified using Life ECO Thermal Cycler (Bioer Technology, Hangzhou, China) according to the manufacturer’s instructions. Autosomal STR genotyping was performed using the 3130 Genetic Analyzer system (Thermo Fisher Scientific). The genotyping results were analysed using GeneMapperTM v3.0 software (Thermo Fisher Scientific).

Library preparation and NGS

One microliter DNA extract from cell-free maternal plasma was amplified using the barcoding primers of the following 12 Y-chromosome mini-STR loci: DYS439, DYS437, DYS643, DYS393, DYS570, DYS392, DYS549, DYS460, DYS458, DYS576, DYS438 and DYS533 (S1 Table). The 2800M Control DNA was used to construct the library. The DNA library was prepared using KAPA Library Amplification Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Specialized adapters and indexes were added to both ends of barcoding sequences (Fig 1). Targeted amplifications were performed in single-tube reactions on a VeritiTM 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA) with the following cycling conditions: 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 4 min; 72 °C for 60 min. Sample indexing with specialized adapters was performed with the following parameters: 95 °C for 5 min; 15 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min. The quantity and purity of the DNA libraries were assessed by SDS-PAGE. Subsequently, the adapter-ligated templates were purified using MagPure A3 XP beads (Angen Biotech). The libraries were quantified with KAPA Library Quant DNA Standards & Primer Premix Kit (Illumina) using the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Each DNA library was normalized to 2nM and pooled in equal volumes. Finally, the DNA libraries were diluted to the concentration of 10pM and sequenced by paired-end 150 bp reads on the NextSeq 500 system (Illumina) (Fig 1).

Fig 1

Schematic representation of library preparation and NGS.

Data for D3S1358 is shown as a representative example. (a) Amplification of the target sequence with barcoding primers. (b) Second PCR amplification adding specialized adapters and indexes to both ends. (c) Purification of the amplicon and pooling multiple libraries together. (d) Linearization of DNA libraries. (e) Loading DNA libraries onto the flow cell. (f) Bridge PCR amplification. (g) Cluster generation. (h) Paired-end sequencing. (i) Base calling, alignment and data analysis.

Data analysis

Quality control (QC) analysis of raw FASTQ data was performed using FastQC software. The FASTQ reads were mapped against reference sequences from GenBank to determine the mini-STR haplotypes of plasma DNA. The sequence and length variants per sample per locus were compiled and counted. The signal noise and stutter ratio of each locus was evaluated independently. The mini-STR haplotyping results of cffDNA were validated by that of paternal genomic DNA (Fig 2).

Fig 2

Flow chart for experimental procedures and data analysis.

Calculation of paternity testing parameters

The combined paternity index (CPI) was calculated according to Eq (1) derived from Rolf’s equation [20] where fs is the frequency of the son’s haplotype, μ is the mutation rate, n is the total number of Y-STR haplotypes, and m is the number of loci where the paternal and filial haplotypes differ. Probability of paternity was calculated by Eq (2) [21]. The CPI and probability of paternity rely on the haplotype distribution in the local population. The haplotype frequencies of the DYS392, DYS393, DYS438, DYS439, DYS437 and DYS458 loci were obtained from Guo’s report [22] on the population genetics of Y-STR loci in the Liaoning population, which is the only local genetic information available at the Y-Chromosome STR Haplotype Reference Database (YHRD) (https://yhrd.org/). Given the limited information on the Y-STR loci haplotypes in the Liaoning population, only 6 out of the 12 loci tested in our study could be used for estimating haplotype frequencies. For example, one of 14 Y-haplotypes with 6 loci in our study matches two of the 838 Liaoning haplotypes available at the YHRD, that results in a haplotype frequency of 0.003521 ((1+2)/(14+838)).

Then, CPI and probability of paternity can be calculated based on this haplotype frequency. According to Eq (1), m = 0 and n = 1 were used in cases without mutation, . For the case with a mutation, the mutation rate μ can be calculated from YHRD. According to Eq (1), m = 1 was used, and .

Results

Paternity validation

Paternity testing with mother, alleged father and infant was performed using commercial autosomal STR genotyping kits by CE, which confirmed the biological relationship between the fathers and infants in all 24 family cases.

STR haplotype-match analysis between cffDNA and paternal genomic DNA

The quantities of the 24 cell-free fetal DNA extracts are shown in S2 Table. Plasma DNA samples were sequenced on the Illumina NextSeq 500 platform, and the number of reads per sample per locus is shown in Fig 3. The average number of the sequence reads for each sample was 283535 (range 84732–819334). The target sequence length of all 12 mini-STR loci was less than 150 bp, of which only 3 STR loci were shorter than 150 bp as per CE (Fig 4). The Y-chromosome mini-STR haplotyping results of 14 male cffDNA samples were obtained without missing loci with NGS. The alleles of cffDNA and paternal genomic DNA were matched in 13 cases, whereas one mismatched allele (14→15) was detected at DYS393 in one case. Given the positive paternity identification, the mismatched allele at DYS393 was considered a mutation. No allele was detected in the 10 female cffDNA samples (S3 Table).

Fig 3

The NGS sequencing reads per sample per locus of plasma DNA (range: 1068–140045 reads).

Fig 4

Overview of target sequence length for all STR loci as per NGS and CE.

Target sequence indicates the PCR amplicon without barcoding and adapter primers.

The probability of paternity using cffDNA was 98.2699–99.8828% for the cases without mutation. The mutation rate of DYS393 is 0.00123 according to YHRD, and taking that into account, the CPI and probability of paternity for case No. 14 were 0.1747 and 14.8719% respectively (Table 1).

Table 1

The paternity testing parameters using cffDNA in the 14 cases with male fetuses.

Case No.	Haplotype frequency	CPI	Probability of paternity
1	0.001174	852	99.8828%
2	0.017606	56.8	98.2699%
3	0.001174	852	99.8828%
4	0.003521	284	99.6491%
5	0.005869	170.4	99.4166%
6	0.001174	852	99.8828%
7	0.003521	284	99.6491%
8	0.001174	852	99.8828%
9	0.001174	852	99.8828%
10	0.003521	284	99.6491%
11	0.001174	852	99.8828%
12	0.001174	852	99.8828%
13	0.002347	426	99.7658%
14a	0.003521	0.1747	14.8719%

aThe case with a mutation at DYS393

Discussion

Capillary Electrophoresis is the routine technique used for STR typing, and is widely used for individual and paternity tests in forensic investigations [23, 24]. NGS is a supplementary tool for forensic genetics owing to the large amount of genetic information that it can provide and process, high throughput function and low costs [25, 26]. Since the length of cffDNA in the maternal plasma is approximately 145-201bp, only short amplicons are available for fetal DNA analysis [9, 27]. Amplicons with similar length must be labelled with different fluorescent markers for CE, which limits the number of loci that can be multiplexed together. In contrast, NGS uses barcodes and index adapters that precludes the need for size separation between amplicons. As a result, numerous loci and multiple samples can be simultaneously analyzed in a single reaction. The mini-STRs were redesigned with primers flanking the repeat region to reduce the amplicon size for small DNA fragment detection [17]. Compared to the commercial kits using CE with amplicon length of 79–430 bp, the mini-STR amplicon lengths analyzed by NGS in our study were all less than 150 bp (Fig 4). Several reports have been published on SNP-based prenatal paternity testing combined with microarrays [28]. Since it relies on the differences in paternally versus maternally inherited SNP alleles, millions of SNPs have to be analyzed due to low polymorphism information content (PIC) leading to statistics with lower power. In addition, the routine use of SNPs in forensics is controversial since some SNPs may be present in the coding regions with bioethical implications stemming from confidentiality and privacy concerns since they may disclose sensitive information or are modified due to disease [29]. In contrast, STRs are widely used for routine forensic applications, and only a set of 12 STR loci is sufficient for paternity testing [30, 31]. Therefore, given the limitations of CE and SNP-based NGS, mini-STR-based NGS with high testing capacity and high PIC is a better choice for NIPPT. According to the guidelines of International Society for Forensic Genetics (ISFG), CPI and probability of paternity should be calculated in paternity testing [32, 33]. Due to strong linkage disequilibrium, the multiplying of single locus allele frequencies cannot be used for Y-STR CPI calculation. Instead, Y-STR-based paternity testing relies on complete haplotype frequencies [12]. In 7 of the 14 cases, the fetal haplotype were not detected in the 838 Liaoning haplotypes reported by Guo et al [22]. According to the ISFG algorithm [14], the haplotype frequency of these cases was 0.0012 (1/852), and the CPI and probability of paternity were 852 and 99.8828% respectively. Since most mutations in the STR loci are caused by DNA strand slippage during DNA replication, the Y-STR loci mutate independent of each other [12, 34]. In our study, one cffDNA sample had a mismatched allele (14→15) at DYS393, which was considered a mutation since paternity had been validated by CE. The sequencing result showed that the repetitive [AGAT] motif of fetal and infant allele had an extra repeat compared to the paternal allele. Unlike the autosomal loci, Y-STR haplotyping may lead to false-positive results if the alleged father and child belongs to the same paternal lineage [16]. For example, the fetus may have the same Y-chromosome as his biological father’s brother or even grandfather’s brother. In this case, Y-STR haplotyping is ineffective if the alleged father and biological father share the exact same Y-chromosome. Thus, Y-STR results must be confirmed with autosomal STR markers when the samples can be safely collected from the babies after birth or from aborted embryos. For this reason, Y-STR haplotyping is normally used along with autosomal STR genotyping to determine paternity. However, it is a viable option in forensics for identifying the paternal male relatives of an unknown perpetrator during large-scale voluntary DNA screening, whereas autosomal STR profiling can only trace the close relatives of the perpetrator [12]. In cases of sexual assault, the cffDNA from pregnant victims bearing male fetuses may provide valuable information regarding the DNA sequence of the perpetrator. Furthermore, for pregnant women with more than one sexual partner, NIPPT with Y-chromosome mini-STR can help exclude paternity if the male fetus and alleged father show no match. Although prenatal screening has been widely accepted, NIPPT is fraught with ethical concerns [35]. It is at present illegal in China in order to avoid its misuse in determining sex of the fetus or in establishing paternity. Nevertheless, we recommend that NIPPT with Y-chromosome mini-STR should be used for unwanted pregnancy as the result of sexual assault or for sex selection in case of gene defect and inherited disease. Sex selection can be a double-edged sword, especially among the economically underprivileged population. While preference for male children leads to significant social problems, medical sex selection can preclude the burden of a child with inherited disease. Nevertheless, avoiding non-medical sex selection remains a serious issue globally.

For the cffDNA samples with no Y-chromosome allele, the sex of all the infants had been determined through paternity testing by CE in our study. Thus, the cffDNA samples lacking Y-chromosome were known before NIPPT. However, in order to conduct a more rigorous study design, an internal control like Amelogenin locus should be included to distinguish DNA amplification failure or absence of Y-chromosome.

In summary, we established the proof-a-concept of Y-chromosome mini-STR-based NIPPT through NGS, which showed high accuracy in real cases and identified a mutation at DYS393. CPI and probability of paternity were calculated based on the local Y-STR haplotype frequencies. The discrimination power could be increased with the availability of more information regarding the haplotype. As an alternative of CE and SNP-based NGS, our approach can aid in familial searching, paternity exclusion and sex selection in forensic and medical applications.

Primers and repeat motif of 12 Y-chromosome mini-STR loci without barcoding sequences.

(DOCX)

Click here for additional data file.

Quantities of the 24 cffDNA extracts.

(DOCX)

Click here for additional data file.

Qualitative results for cffDNA Y-STR haplotypes and their matching to the infant and paternal genomic DNA haplotypes.

(XLSX)

Click here for additional data file.

31 Aug 2021

PONE-D-21-09048

Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing

PLOS ONE

Dear Dr. Ming Liu ,

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I would suggest that you read this following articles which will be of help to answer the reviewers' comments: 

1) Wang et al. STR polymorphisms of "forensic loci" in the northern Han Chinese population. Journal of Human Genetics July 2003, Volume 48, Issue 7, 337-341

2)  Tamaki et al. Microsatellite typing in a paternity case against a deceased man whose two brothers were available for testing.  Jpn J Leg Med 1996 50(2) 82-86

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Reviewer #1: The manuscript describes a strategy for non-invasive prenatal paternity testing by analyzing the Y-chromosome. The method is useful and should be published following revisions (both major and minor) as indicated below (pages and line refer to the PDF format of the manuscript):

MAJOR COMMENTS

Materials and methods, under “DNA extraction”, page 4

1. For the following statement:

“The quantity and purity of the extracted DNA were determined by sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with Tanon 1600 Gel Imaging System (Shanghai Tanon Science & Technology, Shanghai, China) and NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA) respectively”:

provide reference or describe in more detail the methods used. In addition, provide information as to how much of the 60 μl extract was used for SDS-PAGE, for quantitation and for subsequent NGS library preparations.

2. Did the authors use any negative and/or reagent blank controls during DNA extractions and subsequent operations?

Materials and methods, under “Library preparation and NGS”, page 5

3. “DNA extracted from cell-free maternal plasma…”: Provide information as to how many μl of the cell-free DNA extract were used.

4. “…was amplified at the following 12 Y-chromosome mini-STR loci”: Provide the sequence information on the mini-STR primers that were designed for the present assay. In addition, usually barcoding in NGS is used for sample designation, therefore, it is essential that more details should be provided as to the NGS assay with respect to both the samples and the Y-STR loci assayed. More details are required to describe the developed NGS assay.

5. “The quantity and purity of the DNA libraries were assessed by SDS-PAGE.”: Provide reference for the method or more details.

Materials and methods, under “Calculation of paternity testing parameters”, page 6

6. “…the frequency of the son’s haplotype, µ is the mutation rate, n is the total number of Y-STR haplotypes,…”: Provide in this paragraph (i.e. in Materials and methods) the information regarding the use of a mutation rate of 0.00123 for locus DYS393 (indicated on page 8 of the MS under the Results section).

7. Also, another paragraph should be added to indicate that the CPIs were calculated based on 6 loci Y-haplotypes. In other words, the information provided at the beginning of page 10 of the manuscript: “Given the limited information on the Y-STR loci haplotypes in the Liaoning population, only 6 out of the 12 loci tested in our study could be used for calculating CPI and probability of paternity” should be provided in the Materials and Methods section.

Results, pages 6-8

8. The Results section must include (either in Table format or descriptive) information on the quantities of the 24 cell-free fetal DNA extracts as determined by the methods indicated in the Materials and Methods section.

9. As indicated before, a small note, preferably in the Materials and methods section, should indicate that the haplotype frequencies in Table 1 (page 8 of the manuscript) were estimated using the haplotypes for 6 Y-chromosomal loci.

Discussion

10. The inclusion of an internal control to verify that the lack of amplification is due to the absence of the Y-chromosome and not to experimental failure is an important aspect especially in real cases. Perhaps the Amelogenin locus, that has small amplicons could also be included in the assay. Authors to elaborate briefly on this (i.e. how to distinguish failure Vs absence of Y-chromosome) in the discussion section.

References

11. Please go over the references and make sure that authors, journals etc are cited correctly. For example Reference 14 should be corrected to read: Roewer L, Andersen MM, Ballantyne J, Butler JM, Caliebe M, Corach D, ME, Gusmão L, Hou Y, de Knijff P, Parson W, Prinz M, Schneider PM, Taylor D, Vennemann M, Willuweit S (2020) DNA commission of the International Society of Forensic Genetics (ISFG): Recommendations on the interpretation of Y-STR results in forensic analysis. Forensic Science International: Genetics 48: 102308

MINOR COMMENTS/SUGGESTIONS

Abstract

12. Suggestion to revise the sentence, page 1, to read: “The Y-chromosome mini-STR genotypes of all 14 male cffDNA were obtained…”

13. Suggestion to revise the sentence, page 2, to read: “The combined paternity index (CPI) and probability of paternity calculation was based on 6 loci Y-haplotype distributions of a local population.”

Introduction, pages 2

14. Suggestion to revise the sentence, page 2 (last s lines), to read: “Due to its uniparental inheritance, the results of Y-STR typing are interpreted on the basis of haplotype…”

Introduction, page 3

15. Suggestion to revise the sentence, page 3 (1st line), to read: “A major challenge in non-invasive prenatal paternity testing (NIPPT) is the detection of fetal-specific markers of the paternal genetic material…”

Materials and methods, page 4

16. Suggestion to revise the sentence, page 4 (under "DNA extraction", 1st line), to read: “A total of 5 mL peripheral blood was collected from the pregnant women in anticoagulant-treated tubes.”

17. Suggestion to revise the title to read: “Paternity testing by Capillary Electrophoresis (CE)”

Results, page 8

18. Suggestion to revise the title of the Table to read: “Table 1. The paternity testing parameters using cffDNA in the 14 cases with male fetuses”

Discussion, page 9

19. Suggestion to revise the sentence, page 9 (1st sentence - since the beginning of sentence spell out CE) to read: “Capillary Electrophoresis is the routine technique…”

20. Suggestion to revise the sentence, page 9 (lines 2-3), to read: “…genetic information that it can provide and process, high throughput…”

21. Suggestion to revise the sentence, page 9 (line 13), to read: “…millions of SNPs have to be analyzed due to low PIC (polymorphism information content) leading to statistics with lower power. In addition…”

22. Suggestion to revise the sentence, page 9 (lines 14-15), to read: “…SNPs in forensics is controversial since some SNPs may be present in the coding regions with bioethical implications stemming from confidentiality and privacy concerns since they may disclose sensitive information or are modified due to disease [29].”

23. Suggestion to revise the sentence, page 9 (lines 16-17), to read: “Therefore, given the limitations of CE and SNP-based NGS, mini-STR-based NGS with high testing capacity and high PIC is a better choice for NIPPT.”

24. Suggestion to revise the sentence, page 9 (last 2 lines), to read: “Due to strong linkage disequilibrium, the multiplying of single locus allele frequencies cannot be used for Y-STR CPI calculation.”

Discussion, page 10

25. It is recommended to add a small paragraph in Materials and Methods to describe the population that was used to calculate the haplotype frequencies in relation to what is written in the Discussion (page 10; lines 3-4): “In 7 of the 14 cases, the fetal haplotype were not detected in the 838 Liaoning haplotypes reported by Guo et al [22]. According to the ISFG algorithm [14]…”:

26. Suggestion to revise the sentence, page 10 (lines 19-21) to read: “...Y-chromosome mini-STR should be used for unwanted pregnancy as the result of sexual assault or for sex selection in case of gene defect and inherited disease.”

References, page 11

27. Suggestion to change the title to: “References” instead of “Reference”

Reviewer #2: Song et al. propose a proof-of-concept study in which they use massively parallel DNA sequencing to assign paternity to male fetuses in utero. The authors use CPI analysis and compare the alleged father's Y-chromosome STR haplotype to the fetus' haplotype, which is obtained noninvasively from the mother's blood plasma. The manuscript is well-written and the main ideas are clear.

Major comments:

While this study can be considered a valid contribution to science, my main concern is related to how the authors have interpreted their results. I’m not convinced that using only the Y-STR haplotype is sufficient to assign paternity with confidence for two reasons.

First, even though a CPI is calculated to show some degree of uncertainty to the paternity tests performed, we should keep in mind that the Y-STR haplotype represents only one locus in the genome. Thus, this Y-STR paternity analysis would be similar to accepting the use of only one autosomal locus as sufficient to determine paternity with confidence (which can also show greater than 99% CPIs depending on the locus, frequencies and alleles scored).

Second, unlike the autosomal loci, the Y-chromosome is inherited from father to son without recombination. This mode of inheritance creates the potential for several men in the population to share the exact same Y-STR profile (i.e. they belong to the same lineage). For instance, if the paternal grandfather of one of those fetuses had four brothers and each of those brothers (including the grandfather himself) had four sons, and each of those 16 men had two sons, we would have, in this family alone, 52 individuals that would, in theory, match the fetus’ Y-STR profile. This isn’t the case when analyzing autosomal STR profiles (with at least a dozen loci each) due to recombination. In other words, only identical twins are expected to share the same STR profile in the population while several men can (and do) share the same Y-STR haplotype, creating the real potential of false positive results.

For these reasons, unlike it’s implied in the title and throughout manuscript, Y-STR haplotype results alone should not be used to confirm paternity. Instead, it could be used to “exclude” or “not exclude” a male from being a potential father of a fetus. Especially in the forensic setting, where paternity tests can have broad impacts on people's lives, conclusions should always be confirmed with more reliable methods such as genotyping multiple autosomal STR loci.

Minor comments:

1) In the Abstract, where it’s said “The cffDNA genotype was validated by the paternal genotype” I believe the word “genotype” should be replaced by “haplotype” as this sentence seems to be related to the Y-chromosome. I also suggest this wording is checked throughout the manuscript.

2) At the end of the Abstract and the Discussion, the authors mention “sex selection”. This is a very controversial subject due to its ethical implications. And as the authors mention, such practice is forbidden in many countries. Should this potential application be promoted or even discussed in this publication?

3) Materials and Methods (Sample collection): I suggest rewording the first sentence this way: “Peripheral blood samples were collected from 24 pregnant women undergoing prenatal tests, and their male partners, at the Dalian Blood Centre from April 2018 to December 2019”. Dalian Blood *Centre* or Dalian Blood *Center*?

4) Materials and Methods (DNA extraction/library preparation and NGS): Assessing the quantity and purity of DNA extracts and library preparations with SDS-PAGE doesn’t seem to be standard practice. Can you describe a little more how this is done?

5) Figure 2 (flowchart): the boxes “Infant STR genotypes” and “Paternal STR genotypes” appear to be swapped.

Reviewer #3: The study applied a 12 Y-STR multiplex to pre-natal testing of foetal DNA in maternal circulation. Standard statistical paternity testing regimes were applied to confirm paternity. A novel aspect of the work is the use of NGS sequencing to genotype the STRs. However, no primer details or sequence output analysis is given - the Results section is extremely thin, as if the authors assume that the readership would not be interested in how well the NGS assay for these 12 Y-STRs worked with such low-level DNA input. The authors do not discuss their choice of Y loci - why select these 12?

No description is given for the extent to which sequence variation could be used and potentially was of value in the paternity analyses made. This was because normal CE was used to type the matched father's DNA, and NGS for the maternal samples, whereas, it would be potentially better to establish how much sequence variation could benefit the paternity statistical analyses in each case. Therefore, the authors lost every opportunity to report in this paper the sensitivity of the system they have developed in terms of sequence coverage and expanded identification discrimination from sequence variants in the chosen loci.

As such, the work was made scientifically, but there is little of novelty or impact in the study (and I realise the paper cannot be rejected on these grounds alone).

Figures 1 and 2 are largely redundant.

An initial text slip suggested an English review would be beneficial, but in fact, the standard of the rest of the paper is fine:

Our proof-a-concept study demonstrated that Y-chromosome mini-STR can be used > Our proof-of-concept study demonstrated that Y-chromosome mini-STRs can be used.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Christopher Phillips

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10 Oct 2021

Dear Dr. Wang,

Thank you very much for giving us an opportunity to revise our manuscript entitled “Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing”. We are supposed to upload the NGS data, including Metadata spreadsheet, Processed data and Raw data to GEO. But more time is needed for us to get Processed data from Microread Genetics, which is the NGS service supplier for us, due to the laboratory technologist who was in charge of our study resigned from Microread Genetics. We apologize that we cannot upload the NGS data to GEO and provide the relevant accession numbers before October 11. May we provide them later?

The comments from the reviewers have been very helpful in improving our paper and designing new experiments. The revised portions in the manuscript are marked in yellow. The responses to the reviewers’ comments are as follows.

Reviewer #1: The manuscript describes a strategy for non-invasive prenatal paternity testing by analyzing the Y-chromosome. The method is useful and should be published following revisions (both major and minor) as indicated below (pages and line refer to the PDF format of the manuscript):

MAJOR COMMENTS

Materials and methods, under “DNA extraction”, page 4

1. For the following statement:

“The quantity and purity of the extracted DNA were determined by sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with Tanon 1600 Gel Imaging System (Shanghai Tanon Science & Technology, Shanghai, China) and NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA) respectively”:

provide reference or describe in more detail the methods used. In addition, provide information as to how much of the 60 μl extract was used for SDS-PAGE, for quantitation and for subsequent NGS library preparations.

Answer: “Presence of contaminants such as RNA and proteins … was estimated on the basis of a molecular weight marker band.” has been added under “DNA extraction”.

“1 µL” has been added in the “The quantity and purity of the 1µL extracted DNA were determined by sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE)…”.

2. Did the authors use any negative and/or reagent blank controls during DNA extractions and subsequent operations?

Materials and methods, under “Library preparation and NGS”, page 5

Answer: “The 2800M Control DNA (Promega, Madison, WI, USA) was used as the positive control, and nuclease-free water was used as the PCR blank.” has been added under “DNA extraction”

“The 2800M Control DNA was used to construct the library.” has been added under “Library preparation and NGS”.

3. “DNA extracted from cell-free maternal plasma…”: Provide information as to how many μl of the cell-free DNA extract were used.

Answer: It has been changed to “1µL of DNA extract from cell-free maternal plasma …”

4. “…was amplified at the following 12 Y-chromosome mini-STR loci”: Provide the sequence information on the mini-STR primers that were designed for the present assay. In addition, usually barcoding in NGS is used for sample designation, therefore, it is essential that more details should be provided as to the NGS assay with respect to both the samples and the Y-STR loci assayed. More details are required to describe the developed NGS assay.

Answer: The sequence information of mini-STR primers was attached in the S1 Table.

More details about NGS assay have been added in Figure 1.

In the manuscript, “which mainly includes addition of specialized adapters and indexes to both ends of barcoding sequences (Fig 1) … 15 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min.” has been added under “Library preparation and NGS”.

5. “The quantity and purity of the DNA libraries were assessed by SDS-PAGE.”: Provide reference for the method or more details.

Answer: The DNA libraries were constructed by two-step PCR and purified using MagPure A3 XP beads. We assessed the quantity and purity of libraries before and after the purification by SDS-PAGE and Qubit fluorometer respectively. For more details, “Each DNA library was normalized to 2nM and pooled in equal volumes. Finally, the DNA libraries were diluted to the concentration of 10pM and…” has been added under “Library preparation and NGS”.

Materials and methods, under “Calculation of paternity testing parameters”, page 6

6. “…the frequency of the son’s haplotype, µ is the mutation rate, n is the total number of Y-STR haplotypes,…”: Provide in this paragraph (i.e. in Materials and methods) the information regarding the use of a mutation rate of 0.00123 for locus DYS393 (indicated on page 8 of the MS under the Results section).

Answer: “For the case with a mutation, the mutation rate µ can be obtained from YHRD. According to equation (1), m=1 was used, and CPI=(1/2µ)/fs.” has been added under “Calculation of paternity testing parameters”.

7. Also, another paragraph should be added to indicate that the CPIs were calculated based on 6 loci Y-haplotypes. In other words, the information provided at the beginning of page 10 of the manuscript: “Given the limited information on the Y-STR loci haplotypes in the Liaoning population, only 6 out of the 12 loci tested in our study could be used for calculating CPI and probability of paternity” should be provided in the Materials and Methods section.

Answer: The paragraphs “The CPI and probability of paternity calculation rely on the haplotype distribution in the local population … were used in cases without mutation, CPI=1/fs.” has been added under “Calculation of paternity testing parameters”.

Results, pages 6-8

8. The Results section must include (either in Table format or descriptive) information on the quantities of the 24 cell-free fetal DNA extracts as determined by the methods indicated in the Materials and Methods section.

Answer: “The quantities of the 24 cell-free fetal DNA extracts were shown in S2 Table.” has been added under “STR genotype-match analysis between cffDNA and paternal genomic DNA”.

9. As indicated before, a small note, preferably in the Materials and methods section, should indicate that the haplotype frequencies in Table 1 (page 8 of the manuscript) were estimated using the haplotypes for 6 Y-chromosomal loci.

Answer: “Given the limited information on the Y-STR loci haplotypes in the Liaoning population, only 6 out of the 12 loci tested in our study could be used for estimating haplotype frequencies, calculating CPI and probability of paternity.” has been added under “Calculation of paternity testing parameters”.

Discussion

10. The inclusion of an internal control to verify that the lack of amplification is due to the absence of the Y-chromosome and not to experimental failure is an important aspect especially in real cases. Perhaps the Amelogenin locus, that has small amplicons could also be included in the assay. Authors to elaborate briefly on this (i.e. how to distinguish failure Vs absence of Y-chromosome) in the discussion section.

Answer: “For the cffDNA samples with no Y-chromosome allele … distinguish DNA amplification failure or absence of Y-chromosome.” has been added under “Discussion”.

References

11. Please go over the references and make sure that authors, journals etc are cited correctly. For example Reference 14 should be corrected to read: Roewer L, Andersen MM, Ballantyne J, Butler JM, Caliebe M, Corach D, ME, Gusmão L, Hou Y, de Knijff P, Parson W, Prinz M, Schneider PM, Taylor D, Vennemann M, Willuweit S (2020) DNA commission of the International Society of Forensic Genetics (ISFG): Recommendations on the interpretation of Y-STR results in forensic analysis. Forensic Science International: Genetics 48: 102308

Answer: The mistakes in the references have been corrected and highlighted under “Reference”.

MINOR COMMENTS/SUGGESTIONS

Abstract

12. Suggestion to revise the sentence, page 1, to read: “The Y-chromosome mini-STR genotypes of all 14 male cffDNA were obtained…”

Answer: It has been revised in the manuscript.

13. Suggestion to revise the sentence, page 2, to read: “The combined paternity index (CPI) and probability of paternity calculation was based on 6 loci Y-haplotype distributions of a local population.”

Answer: It has been revised in the manuscript.

Introduction, pages 2

14. Suggestion to revise the sentence, page 2 (last s lines), to read: “Due to its uniparental inheritance, the results of Y-STR typing are interpreted on the basis of haplotype…”

Answer: It has been revised in the manuscript.

Introduction, page 3

15. Suggestion to revise the sentence, page 3 (1st line), to read: “A major challenge in non-invasive prenatal paternity testing (NIPPT) is the detection of fetal-specific markers of the paternal genetic material…”

Answer: It has been revised in the manuscript.

Materials and methods, page 4

16. Suggestion to revise the sentence, page 4 (under "DNA extraction", 1st line), to read: “A total of 5 mL peripheral blood was collected from the pregnant women in anticoagulant-treated tubes.”

Answer: It has been revised in the manuscript.

17. Suggestion to revise the title to read: “Paternity testing by Capillary Electrophoresis (CE)”

Answer: It has been revised in the manuscript.

Results, page 8

18. Suggestion to revise the title of the Table to read: “Table 1. The paternity testing parameters using cffDNA in the 14 cases with male fetuses”

Answer: It has been revised in the manuscript.

Discussion, page 9

19. Suggestion to revise the sentence, page 9 (1st sentence - since the beginning of sentence spell out CE) to read: “Capillary Electrophoresis is the routine technique…”

Answer: It has been revised in the manuscript.

20. Suggestion to revise the sentence, page 9 (lines 2-3), to read: “…genetic information that it can provide and process, high throughput…”

Answer: It has been revised in the manuscript.

21. Suggestion to revise the sentence, page 9 (line 13), to read: “…millions of SNPs have to be analyzed due to low PIC (polymorphism information content) leading to statistics with lower power. In addition…”

Answer: It has been revised in the manuscript.

22. Suggestion to revise the sentence, page 9 (lines 14-15), to read: “…SNPs in forensics is controversial since some SNPs may be present in the coding regions with bioethical implications stemming from confidentiality and privacy concerns since they may disclose sensitive information or are modified due to disease [29].”

Answer: It has been revised in the manuscript.

23. Suggestion to revise the sentence, page 9 (lines 16-17), to read: “Therefore, given the limitations of CE and SNP-based NGS, mini-STR-based NGS with high testing capacity and high PIC is a better choice for NIPPT.”

Answer: It has been revised in the manuscript.

24. Suggestion to revise the sentence, page 9 (last 2 lines), to read: “Due to strong linkage disequilibrium, the multiplying of single locus allele frequencies cannot be used for Y-STR CPI calculation.”

Answer: It has been revised in the manuscript.

Discussion, page 10

25. It is recommended to add a small paragraph in Materials and Methods to describe the population that was used to calculate the haplotype frequencies in relation to what is written in the Discussion (page 10; lines 3-4): “In 7 of the 14 cases, the fetal haplotype were not detected in the 838 Liaoning haplotypes reported by Guo et al [22]. According to the ISFG algorithm [14]…”:

Answer: “For example, one of 14 Y-haplotypes with 6 loci in our study matches two of the 838 Liaoning haplotypes available at the YHRD, that results in a haplotype frequency of 0.003521 ((1+2)/(14+838)). Then, CPI and probability of paternity could be calculated based on this haplotype frequency.” has been added under “Calculation of paternity testing parameters”.

26. Suggestion to revise the sentence, page 10 (lines 19-21) to read: “...Y-chromosome mini-STR should be used for unwanted pregnancy as the result of sexual assault or for sex selection in case of gene defect and inherited disease.”

Answer: It has been revised in the manuscript.

References, page 11

27. Suggestion to change the title to: “References” instead of “Reference”

Answer: It has been revised in the manuscript.

Reviewer #2: Song et al. propose a proof-of-concept study in which they use massively parallel DNA sequencing to assign paternity to male fetuses in utero. The authors use CPI analysis and compare the alleged father's Y-chromosome STR haplotype to the fetus' haplotype, which is obtained noninvasively from the mother's blood plasma. The manuscript is well-written and the main ideas are clear.

Major comments:

While this study can be considered a valid contribution to science, my main concern is related to how the authors have interpreted their results. I’m not convinced that using only the Y-STR haplotype is sufficient to assign paternity with confidence for two reasons.

First, even though a CPI is calculated to show some degree of uncertainty to the paternity tests performed, we should keep in mind that the Y-STR haplotype represents only one locus in the genome. Thus, this Y-STR paternity analysis would be similar to accepting the use of only one autosomal locus as sufficient to determine paternity with confidence (which can also show greater than 99% CPIs depending on the locus, frequencies and alleles scored).

Second, unlike the autosomal loci, the Y-chromosome is inherited from father to son without recombination. This mode of inheritance creates the potential for several men in the population to share the exact same Y-STR profile (i.e. they belong to the same lineage). For instance, if the paternal grandfather of one of those fetuses had four brothers and each of those brothers (including the grandfather himself) had four sons, and each of those 16 men had two sons, we would have, in this family alone, 52 individuals that would, in theory, match the fetus’ Y-STR profile. This isn’t the case when analyzing autosomal STR profiles (with at least a dozen loci each) due to recombination. In other words, only identical twins are expected to share the same STR profile in the population while several men can (and do) share the same Y-STR haplotype, creating the real potential of false positive results.

For these reasons, unlike it’s implied in the title and throughout manuscript, Y-STR haplotype results alone should not be used to confirm paternity. Instead, it could be used to “exclude” or “not exclude” a male from being a potential father of a fetus. Especially in the forensic setting, where paternity tests can have broad impacts on people's lives, conclusions should always be confirmed with more reliable methods such as genotyping multiple autosomal STR loci.

Minor comments:

1) In the Abstract, where it’s said “The cffDNA genotype was validated by the paternal genotype” I believe the word “genotype” should be replaced by “haplotype” as this sentence seems to be related to the Y-chromosome. I also suggest this wording is checked throughout the manuscript.

Answer: Thanks for your suggestion. All the words“genotype” have been revised in the manuscript.

2) At the end of the Abstract and the Discussion, the authors mention “sex selection”. This is a very controversial subject due to its ethical implications. And as the authors mention, such practice is forbidden in many countries. Should this potential application be promoted or even discussed in this publication?

Answer: We have discussed more about sex selection in the manuscript. “Sex selection can be a double-edged sword … avoiding non-medical sex selection remains a serious issue globally.” has been added under “Discussion”.

3) Materials and Methods (Sample collection): I suggest rewording the first sentence this way: “Peripheral blood samples were collected from 24 pregnant women undergoing prenatal tests, and their male partners, at the Dalian Blood Centre from April 2018 to December 2019”. Dalian Blood *Centre* or Dalian Blood *Center*?

Answer: The sentence has been revised in the manuscript. I think both “Centre” and “Center” are okay.

4) Materials and Methods (DNA extraction/library preparation and NGS): Assessing the quantity and purity of DNA extracts and library preparations with SDS-PAGE doesn’t seem to be standard practice. Can you describe a little more how this is done?

Answer: “Presence of contaminants such as RNA and proteins … was estimated on the basis of a molecular weight marker band.” has been added under “DNA extraction”.

5) Figure 2 (flowchart): the boxes “Infant STR genotypes” and “Paternal STR genotypes” appear to be swapped.

Answer: The revised figure 2 has been attached.

Reviewer #3: The study applied a 12 Y-STR multiplex to pre-natal testing of foetal DNA in maternal circulation. Standard statistical paternity testing regimes were applied to confirm paternity. A novel aspect of the work is the use of NGS sequencing to genotype the STRs. However, no primer details or sequence output analysis is given - the Results section is extremely thin, as if the authors assume that the readership would not be interested in how well the NGS assay for these 12 Y-STRs worked with such low-level DNA input. The authors do not discuss their choice of Y loci - why select these 12?

Answer: Thanks for your suggestion. The primer sequences have been provided in S1 Table.

We chose these 12 Y loci due to their superior multiplex amplification compared to the others.

No description is given for the extent to which sequence variation could be used and potentially was of value in the paternity analyses made. This was because normal CE was used to type the matched father's DNA, and NGS for the maternal samples, whereas, it would be potentially better to establish how much sequence variation could benefit the paternity statistical analyses in each case. Therefore, the authors lost every opportunity to report in this paper the sensitivity of the system they have developed in terms of sequence coverage and expanded identification discrimination from sequence variants in the chosen loci.

Answer: We appreciate your comment about extending the study to sequence variation, which will be dealt in a subsequent study.

As such, the work was made scientifically, but there is little of novelty or impact in the study (and I realise the paper cannot be rejected on these grounds alone).

Figures 1 and 2 are largely redundant.

Answer: Figures 1 and 2 were exported from Adobe Illustrator Artwork and Microsoft Visio with the highest resolution.

An initial text slip suggested an English review would be beneficial, but in fact, the standard of the rest of the paper is fine:

Our proof-a-concept study demonstrated that Y-chromosome mini-STR can be used > Our proof-of-concept study demonstrated that Y-chromosome mini-STRs can be used.

Answer: The word “proof-a-concept” has been changed to “proof-of-concept” under “Abstract”.

Submitted filename: Response to Reviewer.docx

Click here for additional data file.

15 Nov 2021

26 Nov 2021

Dear Dr. Chan,

Thank you for giving us another chance to revise our manuscript entitled “Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing”. We appreciate the constructive criticism from the reviewers, and have made the necessary changes in the rebuttal letter and manuscript. The revised portion in the manuscript is marked in yellow. The responses to the reviewers’ comments are as follows.

Reviewer #2: Dear colleagues:

In this current version, I believe that Song et al. have addressed most of the criticisms raised by the reviewers in the first submission. However, I'm still VERY CONCERNED about the fact that the authors are proposing a paternity test based exclusivelly on Y-STR haplotypes. In fact, my major comments, which I copy again below, were not responded by the authors.

I would like to stress that unlike autosomal STR genotypes, IDENTICAL Y-STR haplotypes are found in the population because the Y-chromosome is passed on from father to son without recombination. Thus, I strongly suggest that the authors at least acknowledge in the manuscript the possibility of false-positive results due to the inheriteance mode of Y-STR haplotypes. And ideally, the authors could also recommend that the Y-STR results must be confirmed with autosomal STR markers when a sample can be safely collected from the fetus/baby (e.g. after birth or miscarriage).

FROM PREVIOUS REVIEW:

First, even though a CPI is calculated to show some degree of uncertainty to the paternity tests performed, we should keep in mind that the Y-STR haplotype represents only one locus in the genome. Thus, this Y-STR paternity analysis would be similar to accepting the use of only one autosomal locus as sufficient to determine paternity with confidence (which can also show greater than 99% CPIs depending on the locus, frequencies and alleles scored).

Second, unlike the autosomal loci, the Y-chromosome is inherited from father to son without recombination. This mode of inheritance creates the potential for several men in the population to share the exact same Y-STR profile (i.e. they belong to the same lineage). For instance, if the paternal grandfather of one of those fetuses had four brothers and each of those brothers (including the grandfather himself) had four sons, and each of those 16 men had two sons, we would have, in this family alone, 52 individuals that would, in theory, match the fetus’ Y-STR profile. This isn’t the case when analyzing autosomal STR profiles (with at least a dozen loci each) due to recombination. In other words, only identical twins are expected to share the same STR profile in the population while several men can (and do) share the same Y-STR haplotype, creating the real potential of false positive results.

For these reasons, unlike it’s implied in the title and throughout manuscript, Y-STR haplotype results alone should not be used to confirm paternity. Instead, it could be used to “exclude” or “not exclude” a male from being a potential father of a fetus. Especially in the forensic setting, where paternity tests can have broad impacts on people's lives, conclusions should always be confirmed with more reliable methods such as genotyping multiple autosomal STR loci.

Answer: We apologize for not responding your major comments last time. “Unlike the autosomal loci … autosomal STR genotyping to determine paternity.” has been added in the manuscript.

Reviewer #3: All required clarifications and explanations in the text have been made. The description of the figures as 'redundant' was meant to signify that they served no purpose and could be excluded - but their retention is not a problem. I understand the underlying NGS data will be made available in due course, even thought the authors cannot meet this obligation at the moment.

Answer: Thanks for the suggestion. If possible, I hope the description of the figures can be retained. In addition, NGS data has been uploaded to GEO, and the relevant accession number is GSE186434.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

21 Mar 2022

Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing

PONE-D-21-09048R2

Dear Dr. Liu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Please kindly take note the following comments from Reviewer #2 when revising your manuscript:

1) Abstract - This sentence my be missing a period: "The cffDNA haplotype was validated by the paternal haplotype The paternity testing parameters were attributed to each case quantitatively."

2) Figure 2 (and throughout the manuscript) - In some cases it's unclear when the authors are talking about Y-STR HAPLOTYPES or AUTOSOMAL STR GENOTYPES since they did both (e.g. Fig. 2). To better distinguish those, I suggest that when talking about Y-STRs the word "haplotype" is used and when talking about autosomal STRs, the word "genotype" or the phrase "autosomal genotype" is used.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kelvin Yuen-Kwong CHAN, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Abstract - This sentence my be missing a period: "The cffDNA haplotype was validated by the paternal haplotype The paternity testing parameters were attributed to each case quantitatively."

Figure 2 (and throughout the manuscript) - In some cases it's unclear when the authors are talking about Y-STR HAPLOTYPES or AUTOSOMAL STR GENOTYPES since they did both (e.g. Fig. 2). To better distinguish those, I suggest that when talking about Y-STRs the word "haplotype" is used and when talking about autosomal STRs, the word "genotype" or the phrase "autosomal genotype" is used.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

23 Mar 2022

PONE-D-21-09048R2

Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing

Dear Dr. Liu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Kelvin Yuen-Kwong CHAN

Academic Editor

PLOS ONE