Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis

Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design.

Other methods to sex determination have been proposed such as anthropometric measurements of the limbs [36][37][38][39], hands [40,41], and from length of index and ring finger, and the index and ring finger ratio [42][43][44][45]. Nevertheless, it has been reported that 100% of successful sex determinations by osteological measurements only occur when the skeleton is from an adult, it is complete, it is in good condition of preservation, and the morphometric variability in the population to which it belongs is known [46][47][48].
Advances in the field of molecular genetics has provided more sensitive methods for sex determination, such as the polymerase chain reaction (PCR) that allow amplification of single molecules of target DNA to analytical quantities. Biological remains such as hair, bone fragments, or teeth, usually contain some amounts of degraded DNA [48][49][50][51][52][53][54][55][56][57]; therefore, it is possible to establish an individual sex using genetic test. Recent studies have confirmed that teeth are more refractory to contamination by exogenous DNA than bones, although bones can also be good candidates for analysis under some circumstances [58].
Some proposed methods have been based on the analyses of genetic markers lying in the Y chromosome [59][60][61], or in the use of both X-chromosomal and Y-chromosomal STRs [47]. Furthermore, a new method to sex determination using shotgun sequencing has been reported [62], although it might be too expensive for routine application in a large number of samples. Most of these procedures are not sensitive enough, are timeconsuming, expensive, and require an important amount of sample.
Despite the wide list of molecular methods proposed for sex determination, the method based on the amplification of the human amelogenin gene (AMEL) is the most widely used. This gene, originally sequenced by Nakahori et al [63,64], is found on both chromosomes X and Y (GenBank Accession number M55418 for AMELX-allele and M55419 for AMELY-allele). There are sequence and size divergences between them. AMELXallele has a size of 2872 bp and is located on the Xp22.1-Xp22.3 region of the X-chromosome, while the human AMELY-allele has a size of 3272 bp and is located on the Yp11.2 region of the Ychromosome. Male individuals have two different AMEL alleles, one located on chromosome X and one on the Y chromosome, while female individuals have two identical AMEL alleles located on chromosome X [63][64][65][66][67][68][69][70][71][72].
Haas-Rochholz and Weiler [73] reported a total of 19 regions of absolute homology that vary in size from 22 to 80 bp; 5 deletions of 1 to 6 bp located on AmelX-allele; and 5 deletions of 1 to 183 bp located on AmelY-allele thus allowing sex determination by PCR products generated from AMEL-X and AMEL-Y alleles [49,51,52,63,64,67,68,[74][75][76]. The most conventionally used is the 6 bp AMELX-allele specific deletion within intron 1, resulting in 106 and 112 bp PCR products from the X-and Y-alleles, respectively.
However, DNA extracted from less well preserved or older archaeological samples may show higher DNA fragmentation, hampering reproducibility or yielding spurious results [77]. For this reason some researchers have attempted to generate PCR fragments smaller than those described by Sullivan et al. [51] but they are still ineffective for heavily degraded samples [73] and can present methodological difficulties because the analysis also involves post-PCR processes. In other instances, the new methods have not been evaluated for degraded or ancient samples [78,79].
A new strategy developed to sex determination involves the implementation of High Resolution Melting curve analysis (HRM) of PCR products after real-time PCR amplification [80]. This method avoids crossing-over contamination originated from any processing or electrophoretic separations after PCR because the analyses are carried out in the same instrument without exposure of amplicons to the environment (i.e. it does not require any processing or separations after real-time PCR). Furthermore, it allows detection of nonspecific fragments amplified during the PCR step [80][81][82][83]. Therefore, melting-curve analysis is a convenient method, especially in highly degraded samples, because it is possible to detect a simple mutation in a small amplified DNA fragment, avoiding at the same time further contamination risks. This also allows high-throughput analysis of a large number of samples to be carried out in parallel.
Recently, HRM analysis based on real-time PCR using SYBR Green as a DNA-binding dye has been reported [83]. Differential melting curves were produced by a 3-bp deletion on the Xchromosome corresponding to 70 or 73-bp PCR products. The inefficiency of this dye has also been reported: there is a tendency for it to inhibit PCR, promote mispriming, and the relatively instability of the dye, especially under the alkaline conditions in which it is stored [84][85][86]. Other more important adverse effect of SYBR Green is that it can produce unreliable DNA melting curve data due to the so-called ''dye redistribution'' phenomenon [87].
Despite the advantages of PCR-based methods, it has been reported that DNA analysis of degraded or ancient samples can present technical difficulties, such as drop-out phenomena observed mainly in the AMELY-allele in male templates with poor amount of DNA [88][89][90][91][92]. So far, this situation has been reported only in males with a no-native American origin [93,94,95]. According to Kim et al., [92], the most reliable way to avoid the allelic drop-out problem is to replicate PCR results.
We developed a new robust method based on real-time PCR amplification of smaller fragments (61 and 64 bp) of the third exon of the amelogenin gene covering a 3-bp deletion of the AMELXallele, followed by HRM analysis using EvaGreen as the DNAbinding dye. Our method is rapid, cost-effective, sensitive, and it has proven to be useful in samples showing highly degraded DNA. We present here primers and conditions for the analysis and the results of cross-verification with several controls and methodological comparisons.

Sampling and Ethics Statement
The study population came from Teopancazco, a multiethnic neighborhood center located in the southern part of

Genetic analysis
1.-Contamination control. All analyses were carried out under strict conditions for the analysis of ancient DNA, in a dedicated ultraclean room, positively pressurized with filtered and UV-irradiated air, with separation of pre-and post-PCR areas. Laboratory equipments were treated with 30% bleach for DNA contamination removal. The use of disposable filter-plugged pipette tips, tubes, protective cloths, hair covers, laboratory gloves, surgical masks, glasses, and shoes protectors to prevent contaminations was mandatory. Solutions and buffers were irradiated with ultraviolet light. Negative extraction controls and negative PCR controls were always employed.
2.-DNA extraction. Bone and teeth samples were processed in a type II B2 biological security hood, located inside the ultraclean room. The outer surface of the bone or teeth was UVirradiated during 15 minutes and a dental drill was used to remove 0.1 g of powdered material. Powdered samples were added to 5 ml of extraction buffer (0.5 M Tris-HCl pH 8.0-8.5, 0.5 M EDTA pH 8.0, 0.5% SDS and 0.5 mg Proteinase K) and incubated at 37uC for 24 hrs, followed by phenol/chloroform DNA extraction [96]. Aliquots of crude DNA extracts were analyzed with an Agilent 2100 Bioanalyzer Expert High Sensitivity DNA Assay to quantify and assess the quality of extracted DNA.
The fresh blood samples (3 ml) were collected in EDTA vacutainer tubes. Total genomic DNA was isolated using a standard non-organic method and diluted to obtain a working concentration of 2.5 ng/ml. All blood samples were processed in a post-PCR laboratory. The LightCycler 480 Instrument protocol included a preincubation step of 10 min at 95uC. The amplification phase comprised 80 cycles of 15 s, 1 m at 56uC, and 30 s at 72uC. After the PCR step, the High-Resolution Melting analysis was performed measuring the drop of fluorescence signal under the following conditions: 1 m at 95uC, 1 m at 40uC and an increase from 60uC to 90uC at a rate of 1uC/s. The instrument is capable of capturing a large number of fluorescent data points per change in temperature with high precision in order to generate a melting curve chart of sample fluorescence versus temperature using the LightCycler 480 software Ver. 1.5 (Roche Applied Science). The analysis also displays the first negative derivate of these sample curves resulting in a new graph where the melting temperature of each sample appears as a characteristic peak. AMELX-allele and AMELY-allele amplicons produced from both modern male and female DNA templates were evaluated as positive controls and three negative controls with no DNA were evaluated in each amplification.

4.-Cross-verification of the High Resolution Melting
Analysis by Sanger Sequencing. Cloning of PCR products is useful to assess levels of both contamination and molecular damage in ancient DNA. Also, in our experience, it is not easy to sequence accurately short DNA fragments (,80 bp) by Sanger sequencing, therefore in these cases it is useful to clone the fragments and use vector's primer-binding sites for sequencing. For these reasons, the AMELX-allele and AMELY-allele amplicons generated from positive controls and from sex-unknown samples were cloned using a TOPO TA Cloning kit (with pCR 2.1-TOPO vector) and TOP10 chemical Competent cells (Invitrogen) following manufacturer's instructions. A minimum of 30 colonies were selected from each cloned sample and underwent colony PCR using the M13 primers. Then we sequenced 5 clones per sample that contained the transformed vector with the fragment of interest. DNA sequencing was performed in both directions by dideoxy method using the 3730 xl DNA Analyzer (Applied Biosystems) at the Genomic Services Unit in Langebio, Cinvestav, Mexico.

DNA preservation
Several attempts to determine the sex of these samples with two previously published methods were unsuccessful. These methods targeted amelogenin gene fragments of 106-112 bp [51] and 70-73 bp [83]. This was a first indication of DNA degradation. Consistent with this, the Bioanalyzer assay showed a high DNA fragmentation pattern, with a main distribution of DNA fragments in the range of 40-100 bp, in all ancient samples analyzed ( Figure  S1). We therefore considered these samples to be heavily degraded and that represented ideal cases to test our HRM method, which targets smaller amelogenine gene fragments.

Sex determination by High Resolution Melting Analysis
This analysis is based on the melting temperature (Tm) difference of the amplified AMELX-allele and AMELY-allele fragments (61 bp for the AMELX-allele and 64 bp for the AMELY-allele) produced by a 3-bp deletion on the AMELXallele. After software processing, the curves obtained from male and female individuals showed a clear difference in shape (Figure 1), allowing thus sex identification.
The accuracy of the assay was examined by using a set of 6 control samples of known sex. To assess reproducibility, three replicates of each sample were analyzed. No discrepancy was observed between sex determinations by HRM and the previously known sex status in all control samples and their replicas. Regarding sex-unknown archaeological samples, this method was efficient in all the individuals examined in spite of the observed pattern of DNA degradation. However, for one of the individuals only two replicates were done, because the DNA extract was not enough for the three replicates due to its use in previous experiments.

Cross-verification of the High Resolution Melting Analysis
The accuracy of sex determination by HRM analysis was verified by contrasting results with those obtained with other three methods. The first one was the sequencing of 5 positive clones harboring fragments from the AMELX-allele and AMELY-allele amplified from samples of known sex. The sequences obtained demonstrated the 3-bp insertion on the AMELY-allele in the male samples ( Figure 2).
A second confirmation of the HRM analysis was conducted by a Bioanalyzer assay, in which AMELX-allele and AMELY-allele amplicons generated by Real-Time PCR from positive controls and from sex-unknown samples were analyzed. The results showed a differential pattern in terms of amplicons length between male and female individuals (Figure 3).
The third cross-verification was conducted by contrasting the HRM data with previously obtained sex determination by osteological measurements in 4 adult individuals recovered from Teopancazco, Teotihuacan (burials 2, 78, 102 and 105). The sex determination by HRM analysis matched with the sex determined by osteological measurements in all the samples analyzed.  Discussion Sex determination in highly degraded samples from human remains has been a challenge in the anthropological and forensic fields. Traditionally, it has been based on the visual examination of the sexual dimorphism present in the majority of human bones and dentition . Nevertheless, these kinds of methods are not appropriate if the skeletons are sub-adult, incomplete or highly damaged [46][47][48]. For this reason, in the last years a large number of methods for sex determination based on molecular biology have been proposed [48][49][50][51][52][53][54][55][56][57]. The most widely used is based on the human amelogenin gene (AMEL) [63][64][65][66][67][68][69][70][71][72] because this analysis can distinguish AMEL alleles from each sexual chromosome due to genetic differences between them, as described previously by Haas-Rochholz and Weiler [73].
At the moment, different primer sets to amplify the amelogenin gene have been reported [49,51,52,63,64,67,68,[74][75][76]. However, the length of the fragments amplified is relatively large which causes this method to be unsuitable for heavily degraded samples. A targeted DNA fragment size of 100 bp might be too long for a successful and reliable analysis in some cases [97,98]; especially considering this is nuclear DNA, more prone to degradation as compared to mitochondrial DNA.
In our study, the ancient samples showed a high DNA fragmentation pattern (between 40-100 bp) as assessed by a Bioanalyzer assay, indicating a general poor DNA preservation. As a result, primer sets published previously [51] were not efficient for sex determination in these samples (data not shown). This prompted us to devise a new method to sex identification analyzing smaller amplicons, suitable for heavily degraded samples. There are other published methods that involve smaller amplicons than ours, but they also involve post-PCR process, such as electrophoresis, Denaturing High Performance Liquid Chromatography (DHPLC) [78], or pyrosequencing [79], which can cause crossing-over contamination, especially in ancient samples.
Here we propose a new method based on the melting-curve analysis reported firstly by Ririe et al., [80]. This method is more adequate for samples with extremely poor preservation, as it is more sensitive, while at the same time it avoids crossing-over contaminations, by not requiring any processing after real-time PCR as in other previously published methods [78,79]. Furthermore, it allows detecting in a small amplified DNA fragment, the 3 bp deletion in the AMEL-X allele.
The use of EvaGreen in our method is relevant because it is stable both under PCR conditions and during routine storage and handling. Furthermore, no PCR interference is produced when used at low concentration, thus permitting a more robust PCR signal as well as a sharper and stronger DNA melt peak in meltingcurve analyses [96,99]. In fact, the melting-curve obtained by our method shows a clear difference in shape between male (2 peaks) and female individuals (single peak) (Figure 1).
Ancient or degraded samples are prone to the allele drop-out phenomenon, consisting in amplification failure of only one of the two alleles in a locus, mainly the largest one [100], indicating an inverse relationship between amplicon fragment and amplification failure. Therefore, a reduction of allele drop-out frequency could be expected by targeting smaller fragment sizes. On the other hand, to circumvent the problem it has been recommended to analyze several replicas of the same sample [92]. Therefore, we conducted the analysis by triplicate in all samples but one. Results from the three replicas were successfully obtained in 16 samples out of 17 samples analyzed. The exception was a sample for which DNA was finished at the second replica. However a consistent result was obtained from the two replicas for this sample.
Our method was cross-verified by three analyses. HRM results and those obtained by (i) sequencing several cloned amplicons from the AMELX-allele and AMELY-allele, (ii) a Bioanalyzer assay, and (iii) previous anthropological data [101], were all concordant. This HRM method was efficient to determine the sex of 16 individuals of unknown sex recovered from Teopancazco, Teotihuacan. Fourteen of them were infants and three samples were from adult individuals with sex reported as no consistent by anthropological data. This illustrates the usefulness of this method to sex identification in samples in which other previously published molecular methods [51,83] failed (see DNA preservation section above).
A variety of methodologies to sex determination have been reported previously, but are not sensitive enough on samples presenting extensive degradation of genetic material, as is the case of Mesoamerican populations subjected to warmer conditions in relation to more northern ancient populations. Our method demonstrated to be efficient, rapid and sensitive enough for sex determination in human remains showing a general poor DNA preservation. Finally, the method can be used in a highthroughput manner to analyze a relatively large number of samples in one run, at a relatively low cost. For example, including replicas and controls, 21 samples can be analyzed in a 96-well PCR plate and 84 samples in a 384-well PCR plate. Figure S1 DNA fragmentation pattern in a sample recovered from Teopancazco. Main distribution of DNA fragments is in the range of 40-100 bp. (TIF)