Performance evaluation of an in-house panel containing 59 autosomal InDels for forensic identification in Chinese Hui and Mongolian groups.

In recent years, a novel multiplex system containing two mini-short tandem repeats, 59 autosomal InDels, two Y-chromosomal InDels, and the Amelogenin gene with all amplicons less than 200 bp has been constructed and validated by ourselves for forensic degration sample, and its forensic application efficiency has been studied in Chinese some populations. Herein, the population genetic polymorphisms of these loci were investigated in Chinese Hui (n = 249) and Mongolian (n = 222) ethnic groups using direct multiplex amplification and capillary electrophoresis platform. The forensic identification efficiencies of this self-developed system were further evaluated in these two groups. And the results showed that the values of the combined power of discrimination were 0.9999999999999999999999999999006 (Hui) and 0.999999999999999999999999999738 (Mongolian), respectively. Moreover, the combined power of exclusion values were 0.99999817 (Hui) and 0.99999779 (Mongolian). The 59 autosomal InDels used in this study exhibited high forensic identification efficiencies in 10 East Asian populations, which was also expected to be a new powerful tool for identifying degraded biological materials in East Asian populations.

Selection and validation of a novel set of specific differential methylation markers and construction of a random forest prediction model for the accurate tissue origin identifications of body fluids involving young and middle-aged group of Chinese Han population.

The identification of tissue origin of body fluid is helpful to the determination of the case nature and the reproduction of the case process. It has been confirmed that tissue-specific differential methylation markers could be used to identify the tissue origins of different body fluids. To select suitable tissue-specific differential methylation markers and establish the efficient typing system which could be applied to the identifications of body fluids in forensic cases involving Chinese Han individuals of young and middle-aged group, a total of 125 body fluids (venous blood, semen, vaginal fluid, saliva, and menstrual blood) were collected from healthy Chinese Han volunteers aged 20-45 years old. After genome-wide explorations of DNA methylation patterns in these five kinds of body fluids based on the Illumina Infinium Methylation EPIC BeadChip, 15 novel body fluid-specific differential CpGs were selected and verified based on the pyrosequencing method. And these identification efficiencies for target body fluids were verified by ROC curves. The pyrosequencing results indicated that the average methylation rates of nine CpGs were consistent with those of DNA methylation chip detection results, and the other five CpGs (except for cg12152558) were still helpful for the tissue origin identifications of target body fluids. Finally, a random forest classification prediction model based on these 14 CpGs was constructed to successfully identify five kinds of body fluids, and the tested accuracy rates all reached 100%.

Comprehensive evaluations of individual discrimination, kinship analysis, genetic relationship exploration and biogeographic origin prediction in Chinese Dongxiang group by a 60-plex DIP panel.

BACKGROUND: Dongxiang group, as an important minority, resides in Gansu province which is located at the northwest China, forensic detection system with more loci needed to be studied to improve the application efficiency of forensic case investigation in this group. METHODS: A 60-plex system including 57 autosomal deletion/insertion polymorphisms (A-DIPs), 2 Y chromosome DIPs (Y-DIPs) and the sex determination locus (Amelogenin) was explored to evaluate the forensic application efficiencies of individual discrimination, kinship analysis and biogeographic origin prediction in Gansu Dongxiang group based on the 60-plex genotype results of 233 unrelated Dongxiang individuals. The 60-plex genotype results of 4582 unrelated individuals from 33 reference populations in five different continents were also collected to analyze the genetic background of Dongxiang group and its genetic relationships with other continental populations. RESULTS: The system showed high individual discrimination power, as the cumulative power of discrimination (CPD), cumulative power of exclusion (CPE) for trio and cumulative match probability (CMP) values were 0.99999999999999999999997297, 0.999980 and 2.7029E- 24, respectively. The system could distinguish 98.12%, 93.78%, 82.18%, 62.35% and 39.32% of full sibling pairs from unrelated individual pairs, when the likelihood ratio (LR) limits were set as 1, 10, 100, 1000 and 10,000 based on the simulated family samples, respectively. Additionally, Dongxiang group had the close genetic distances with populations in East Asia, especially showed the intimate genetic relationships with Chinese Han populations, which were concluded from the genetic affinities and genetic background analyses of Dongxiang group and 33 reference populations. In terms of the effectiveness of biogeographic origin inference, different artificial intelligent algorithms possessed different efficacies. Among them, the random forest (RF) and extreme gradient boosting (XGBoost) algorithm models could accurately predict the biogeographic origins of 99.7% and 90.59% of three and five continental individuals, respectively. CONCLUSION: This 60-plex system had good performance for individual discrimination, kinship analysis and biogeographic origin prediction in Dongxiang group, which could be used as a powerful tool for case investigation.

Forensic efficacy evaluation and genetic structure exploration of the Yunnan Miao group by a multiplex InDel panel.

The aim of the study was to better understand the genetic characteristics of the Miao group in China. Herein, genetic characteristics and forensic application values of 57 autosomal insertion-deletion (InDel) loci were investigated in 210 unrelated healthy individuals from the Chinese Yunnan Miao (YM) group. Meanwhile, the genetic differences in these InDels were compared among the YM group and 26 reference populations. The results of forensic statistical analyses showed that all 57 autosomal InDels were in accordance with the Hardy-Weinberg and linkage equilibria of pairwise loci in the Chinese YM group. Moreover, the combined probability of discrimination and probability of exclusion in the YM group were 0.9999999999999999999999801 and 0.999928, respectively, which indicated that the multiplex amplification including 57 autosomal InDels was suitable for forensic individual identification and paternity testing in the Chinese YM group. In addition, the results of allelic frequency distribution differential analyses, principal component analyses, phylogenetic tree reconstruction, and genetic structure analyses between the Chinese YM group and 26 reference populations revealed that the genetic similarities between the YM group and East Asian populations were more than that between the YM group and other geographical populations. This 57 autosomal InDels system can also effectively distinguish East Asian, European, and African populations.

Insights into AIM-InDel diversities in Yunnan Miao and Hani ethnic groups of China for forensic and population genetic purposes.

BACKGROUND: Ancestry informative markers are regarded as useful tools for inferring the ancestral information of an individual, which have been widely used in the criminal investigations and population genetic studies. Previously, a multiplex amplification panel containing 39 AIM-InDel loci was constructed. This study aims to investigate the genetic polymorphisms of these 39 AIM-InDel loci in Yunnan Hani and Miao ethnic groups, and to uncover their genetic affinities with reference populations based on the AIM-InDel markers. MATERIALS AND METHODS: In this research, 39 AIM-InDel profiles of 203 unrelated Miao individuals and 203 unrelated Hani individuals in Yunnan province of China were acquired. Additionally, we evaluated the genetic polymorphisms of 39 InDel loci in Yunnan Miao and Hani groups. Moreover, the genetic relationships among Yunnan Miao, Hani and reference populations were also clarified based on Nei's genetic distances, pairwise fixation indexes, principal component analyses, phylogenetic analyses, and STRUCTURE analyses. RESULTS: Genetic diversity analyses demonstrated that these InDel loci showed varying degrees of genetic polymorphisms, and could be utilized in forensic identifications in Yunnan Miao and Hani groups. The results of principal component analyses, phylogenetic analyses and Structure analyses revealed that Yunnan Miao and Hani groups had closer genetic relationships with East Asian populations, especially with the populations from Southern China. This research enriched the genetic data of Chinese ethnic minority, and provided ancestral information of Yunnan Miao and Hani groups from the perspective of population genetics.

Multiple genetic analyses for Chinese Hunan Han population via 46 A-STRs.

BACKGROUND: Short tandem repeats (STRs) are important as common genetic markers in forensic identification and population genetics due to their highly polymorphic nature. AIMS: To explore genetic polymorphisms of the Chinese Hunan Han population and further dissect genetic relationships among the Hunan Han and other populations from China. SUBJECTS AND METHODS: In this study, samples of 394 unrelated healthy individuals from the Chinese Hunan Han population were analysed using 46 autosomal-STRs (A-STRs). Thirteen previously reported populations (6378 individuals) from China were subsequently collected for population genetic analyses based on 23 shared A-STRs. RESULTS: In the Hunan Han population, a total of 452 alleles were detected in 46 A-STRs with allelic frequencies spanning from 0.0013 to 0.5571. Except for the Penta D locus in linkage disequilibrium, the combined power of discrimination and the combined power of exclusion for 45 A-STRs in the Hunan Han population were 0.999999999999999999999999999999999999999999999999510314 and 0.999999999999999726596, respectively. Results of interpopulation differentiation, principal component analysis, and phylogenetic relationship analyses uniformly showed that the Hunan Han have closer genetic affinities with Han populations from different Chinese regions and a geographically close ethnic minority group, namely the Hubei Tujia. CONCLUSION: To summarise, these 46 A-STRs showed high polymorphism in the Chinese Hunan Han population for forensic practice.

New Advances, Challenges and Opportunities in Forensic Applications of Microbiomics.

The succession of microbiota is closely associated with several essential factors, including race, sex, health condition, lifestyle, postmortem interval, etc., and it has great potential application value in forensic medicine. This paper summarizes recent studies on the forensic applications of the microbiome, including individual identification, geographical feature identification, origin identification of the tissue or body fluid, and postmortem interval estimation, and introduces the current machine learning algorithms for microbiology research based on next-generation sequencing data. In addition, the current problems facing forensic microbiomics such as the extraction and preservation of samples, construction of standardization and database, ethical review and practical applicability are discussed. Future multi-omics studies are expected to explore micro ecosystems from a comprehensive and dynamic perspective, to promote the development of forensic microbiomics application.

Next generation sequencing of a set of ancestry-informative SNPs: ancestry assignment of three continental populations and estimating ancestry composition for Mongolians.

When traditional short tandem repeat profiling fails to provide valuable information to arrest the criminal, forensic ancestry inference of the biological samples left at the crime scene will probably offer investigative leads and facilitate the investigation process of the case. That is why there are consistent efforts in developing panels for ancestry inference in forensic science. Presently, a 30-plex next generation sequencing-based assay was exploited in this study by assembling well-differentiated single nucleotide polymorphisms for ancestry assignment of unknown individuals from three continental populations (African, European and East Asian). And meanwhile, relatively balanced population-specific differentiation values were maintained to avoid the over-estimation or under-estimation of co-ancestry proportions in individuals with admixed ancestry. The principal component analysis and STRUCTURE analysis of reference populations, test populations and the studied Mongolian group indicated that the novel assay was efficient enough to determine the ancestry origin of an unknown individual from the three continental populations. Besides, ancestry membership proportion estimations for the Mongolian group revealed that a large fraction of the ancestry was contributed by East Asian genetic component (approximately 83.9%), followed by European (approximately 12.6%) and African genetic components (approximately 3.5%), respectively. And next generation sequencing technology applied in this study offers possibility to incorporate more single nucleotide polymorphisms for individual identification and phenotype prediction into the same assay to provide as many as possible investigative clues in the future.

Insights into the genetic characteristics and population structures of Chinese two Tibetan groups using 35 insertion/deletion polymorphic loci.

Studying the genetic structure of each ethnic group is helpful to clarify the genetic background and trace back to the ethnic origin. Tibetan people have lived in the Qinghai-Tibet Plateau (mean elevation over 4500 m) for generations, and have well adapted to the high-altitude environment. Due to the relatively closed geographical environment, Tibetans have preserved their representative physical characteristics and genetic information, thereby become an important research group in human genetics. In this study, genetic characteristics and population structures of two Tibetan groups (Qinghai Tibetans and Tibet Tibetans) were revealed by 35 insertion/deletion polymorphism (DIP) loci, aiming to provide valuable genetic information for population genetic differentiation analyses and forensic identifications. The combined discrimination power, cumulative exclusion probability and combined match probability of the 35 DIP loci in Qinghai Tibetan and Tibet Tibetan groups were 0.9999999999999945, 0.9988, 5.56623 × 10-15; and 0.9999999999999904, 0.9990, 9.69071 × 10-15, respectively, indicating that the panel possessed a strong capability for Tibetan personal identifications. Population differentiations and genetic relationship analyses among the two studied Tibetan groups and other 27 comparison populations were carried out using the Nei's DA genetic distances, population pairwise genetic distances F-statistics (FST), analysis of molecular variance (AMOVA), phylogenetic tree reconstruction, principal component analysis and STRUCTURE methods. Results demonstrated that the most intimate genetic relationships existed in these two Tibetan groups; and genetic similarities between two Tibetan groups and the populations from East Asia were much stronger than that between the Tibetan groups and other geographical populations. Furthermore, forensic ancestral informativeness assessments suggested that several loci could be regarded as ancestry informative markers inferring individual biogeographic origins as well as contributing to forensic anthropology and population genetic researches.

Ancestry inference and admixture component estimations of Chinese Kazak group based on 165 AIM-SNPs via NGS platform.

Predicting the biogeographical ancestries of populations and unknown individuals based on ancestry-informative markers (AIMs) has been widely applied in providing DNA clues to criminal investigations, correcting the factor of population stratification in genome-wide association studies (GWAS), and working as the basis of predicting the externally visible characteristics (EVCs) of individuals. The present study chose Chinese Xinjiang Kazak (XJK) group as research object using a 165 AIM-SNPs panel via next generation sequencing (NGS) technology to reveal its ancestral information and genetic background by referencing the populations' data from 1000 Genomes Phase 3. After the Bonferroni correction, there were no significant deviations at the 165 AIM-SNP loci except two loci with homozygote in the studied XJK group. Ancestry information inference and populations genetic analyses were conducted basing on multiplex statistical methods such as forensic statistical parameter analyses, estimation of the success ratios with cross-validation, population tree, principal component analysis (PCA), and genetic structure analysis. The present results revealed that XJK group had the admixed ancestral components of East Asian and European populations with the ratio of about 62:37.

A set of novel multi-allelic SNPs for forensic application developed through massively parallel sequencing and its examples of population genetic studies.

Massively parallel sequencing (MPS) technology allows to simultaneously type multitudinous molecular genetic markers for many samples in one run with the feature of high detection resolution, and thereby arouses the increasing attention from forensic science. Herein, multiple allelic single nucleotide polymorphisms (multi-allelic SNPs) were screened for personal identification and parentage testing, and then were genotyped using MPS platform. Unrelated individuals of Chinese Mongolian and Kazakh groups were investigated to further estimate forensic effectiveness and applicability of these multi-allelic SNPs. The results of sequencing efficiency estimations and forensic genetic statistical parameters demonstrated that this MPS panel of multi-allelic SNPs was expected to be work for forensic applications. Subsequently, the exploration of population genetic variation patterns among the two investigated groups and other 26 reference populations revealed that these Chinese Mongolian and Kazakh groups had the similar population genetic patterns with the populations from East Asian, but European ancestral composition in the Kazakh group was higher than that in the Mongolian group. Currently, the present results were the preliminary research to scrutinize genetic information of these two ethnic minority groups employing multi-allelic SNPs.

Detection and analysis of the cause of false-tetra-allelic patterns of locus D10S1435 at the sequence level.

A number of artifacts produced in forensic DNA typing make the interpretation more complicated and even lead to typing errors. Here, we reported the cause of false-tetra-allelic patterns of STR locus D10S1435 at the sequence level. To confirm the true genotyping, the sample with four allelic peaks was re-amplified and sequenced. The amplicon sequences of D10S1435, D20S482, D6S1017, and D10S1248 loci were analyzed by software BioXM and RNAstructure. We successfully reproduced the four-peak phenomenon by adding various concentration of magnesium chloride into the loading mixtures to simulate the suboptimal electrophoresis conditions. The false four allelic peaks may be caused by the specific nucleotide sequence of locus D10S1435 which tends to form secondary structures under the suboptimal electrophoresis conditions. The relatively high GC content and extremely uneven distribution give the amplicon a potency to resist complete denaturation at the phase of sample preparation and a tendency to form intra- and intermolecular secondary structures during post-injection.

The validation study of a novel assay with 30 slow and moderate mutation Y-STR markers for criminal investigation and database applications.

The Y chromosome short tandem repeat (Y-STR) haplotyping method has been widely used in forensic applications. However, the existing Y-STR panels are not the ideal tools for criminal investigation and database applications because of their relatively low discriminatory capacity (DC) or high mutation rates. In the present study, the multiplex PCR assay (AGCU Y30) for simultaneous amplification of 30 slowly and moderately mutated Y-STR loci labeled by 6-dye fluorescence was developed and validated. The AGCU Y30 assay was capable of amplification purified DNA from casework and database samples on FTA™ cards in direct amplification module with a 10 µL reaction volume. Furthermore, the genetic diversities and forensic parameters of AGCU Y30 were performed using 719 unrelated male samples, demonstrating its high level of genetic polymorphisms and DC in Nantong Han population. This validation study demonstrated good sensitivity, mixture samples, inhibitor tolerance, precision, and concordance for the AGCU Y30, which is suitable for forensic investigation and database construction.

Developmental validation study of a 24-plex Y-STR direct amplification system for forensic application.

In the present study, validation data for 24 Y-STR loci from the Microreader™ 24Y Direct ID System was presented. Eight Y-STR loci have PCR product sizes with less than 220 bp in this multiplex amplification system, which can better detect degraded DNA samples from a crime scene. Developmental validation studies were conducted following the SWGDAM guidelines and consisted of PCR-based studies, sensitivity testing, species specificity, stability studies, accuracy and reproducibility evaluation, mixture studies, and case-type samples. The genetic diversities and forensic parameters of the 24 Y-STR loci were also investigated in Jiangsu Han population. Results demonstrated that this kit had the characteristics of high detection accuracy, strong species specificity, favorable anti-inhibition effect, and high sensitivity, and the minimum detection amount was 125 pg. When the mixed female template amount was below 3.2 times that of the male, or the male-male mixed ratio did not exceed 1:9, the typing results produced by 24Y Direct System still exhibited a higher discriminating ability for the mixture. The system was compatible with some typical biological samples such as bloodstain, hair, buccal swab, rib cartilage, and nail. The haplotype diversity (HD) and discrimination capacity (DC) of the 24 Y-STR loci were 0.9952 and 0.8500, respectively. The results revealed that the 24 Y-STR loci were highly polymorphic in Jiangsu Han population and could be useful for forensic cases and population genetic studies.

Distinguishing three distinct biogeographic regions with an in-house developed 39-AIM-InDel panel and further admixture proportion estimation for Uyghurs.

In the forensic field, ancestry-informative markers (AIMs) showing remarkable allele frequency discrepancies can be useful in deducing the likely ancestral origin of a person or estimating the ancestry component proportions of an admixed population or individual. Diallelic single nucleotide polymorphisms are genetic markers commonly used for ancestry inference, but the genotyping methods of single nucleotide polymorphisms fail to fulfil the demands of cost-effectiveness and simplicity of experimental manipulation. To overcome the limitations, a 39 ancestry-informative insertion/deletion polymorphism multiplex panel was developed in the present study to perform ancestry assignment of individuals from three distinct biogeographic regions (Africa, Europe, East Asia). And in the panel design, we also attempted to incorporate AIM-insertion/deletion polymorphisms exhibiting allelic frequency differences in Han, Uyghur, and Tibetan populations into the multiplex assay, further expecting to provide valuable information for refining ancestry inference within Chinese populations. Statistical analyses were performed to estimate efficiency of this panel in clustering individuals from three continents mentioned above into their corresponding populations, which indicated the potential of the panel in ancestry inference. Besides, we also estimated the ancestral component proportions of Uyghur group and STRUCTURE analysis revealed that Uyghurs from Urumchi city of northern Xinjiang exhibited a distinctly admixed pattern of East Asian and European ancestry components with a ratio of 49:44, reflecting the relatively higher East Asian ancestry component contribution in the gene pool of the Uyghur group.

Multiple genetic analyses to investigate the polymorphisms of Chinese Mongolian population with an efficient short tandem repeat panel.

AIM: To determine allele frequencies and forensic statistics of 22 autosomal short tandem repeat loci in Chinese Mongolian population. METHODS: Blood specimens were collected from 134 unrelated healthy Mongolian individuals, and 22 short tandem repeat loci were co-amplified and genotyped. Allele frequencies and forensic parameters were calculated, and population genetic differences were analyzed among Mongolian population and other eight Chinese populations: Northern Han, Guangdong Han, Chengdu Han, Xinjiang Hui, Xinjiang Uygur, Hainan Li, Qinghai Tibetan, and Hainan Han. RESULTS: All the loci were in the Hardy-Weinberg equilibrium, and after Bonferroni correction there was no linkage disequilibrium between them. The allele frequencies of these 22 loci were between 0.0037 and 0.3657. This panel had high discriminating power and genetic polymorphism in the Mongolian population, with combined power of discrimination of 0.999999999999999999999999998399 and combined probability of exclusion of 0.9999999999566925. Structure analysis showed no evidence that these nine Chinese populations had different component distribution. However, genetic distance analysis showed significant differences among them (P<0.05). CONCLUSION: The combined application of these 22 loci could be useful for forensic purposes in the Mongolian population. Mongolian population had smaller genetic distances from the populations in northern China (Northern Han, Xinjiang Uygur, and Xinjiang Hui) than from the populations in Hainan province (Hainan Han and Hainan Li populations).

Forensic molecular genetic diversity analysis of Chinese Hui ethnic group based on a novel STR panel.

In present study, the genetic polymorphisms of 22 autosomal short tandem repeat (STR) loci were analyzed in 496 unrelated Chinese Xinjiang Hui individuals. These autosomal STR loci were multiplex amplified and genotyped based on a novel STR panel. There were 246 observed alleles with the allele frequencies ranging from 0.0010 to 0.3609. All polymorphic information content values were higher than 0.7. The combined power of discrimination and the combined probability of exclusion were 0.999999999999999999999999999426766 and 0.999999999860491, respectively. Based on analysis of molecular variance method, genetic differentiation analysis between the Xinjiang Hui and other reported groups were conducted at these 22 loci. The results indicated that there were no significant differences in statistics between Hui group and Northern Han group (including Han groups from Hebei, Henan, Shaanxi provinces), and significant deviations with Southern Han group (including those from Guangdong, Guangxi provinces) at 7 loci, and Uygur group at 10 loci. To sum up, these 22 autosomal STR loci were high genetic polymorphic in Xinjiang Hui group.

Mutability analysis towards 21 STR loci included in the AGCU 21 + 1 kit in Chinese Han population.

To further illustrate the mutation traits of 21 autosomal STR loci involved in the newly developed AGCU 21 + 1 kit, randomly selected 485 trios were focused on. We have previously confirmed the biological father-mother-child relationships of these trios. Then matters of mutation rates, steps, and origins of the 21 STR loci were statically analyzed. Results showed 35 mutation events occurred at 16 STR loci of the panel during 20,370 meiosis, and the locus-specific mutation rates ranged from 0.000 to 1.134% (D3S4529), with an overall mutation rate of 1.72 × 10-3 (95%CI, 1.20-2.39 × 10-3). Mutation origins exhibited slight discrepancy between male and female germlines, 18 of the 35 mutation events were verified to be contributed by fathers and 13 by mothers, with origins of the remaining 4 events hardly to be assigned. Furthermore, 77.14% of the mutations were one-step mutation. Currently, portraits on mutability of the STR loci involved in the AGCU 21 + 1 panel were barely reported. In view of this, we conclude our data will enrich the mutation information of the 21 STR loci and provide valuable suggestions in calculating likelihood ratios for mutation-involved parental testing cases.

Genetic structure and polymorphism analysis of Xinjiang Hui ethnic minority based on 21 STRs.

In the present study, we calculated the allelic frequencies and forensic descriptive parameters of Hui ethnic minority on the basis of 21 short tandem repeat (STR) loci aiming at understanding population structure better and enriching population genetic database. Bloodstain samples of 506 unrelated healthy Hui individuals in Xinjiang Uygur Autonomous Region were collected. Altogether 268 alleles were observed and the allelic frequencies ranged from 0.0010 to 0.5306. The combined power of discrimination and the cumulative probability of exclusion of the 21 STR loci in Hui ethnic minority were 0.9999999999999999999999998697 and 0.9999999968, respectively. Population data obtained manifested that the panel of 21 STR loci could provide robust genetic information for individual identification and paternity testing involved in forensic applications for Huis of Xinjiang Region. Furthermore, the present results of interpopulation differentiations, phylogenetic trees and principal component analysis which were conducted based on the overlapping 16 STR loci revealed that Hui group was genetically close to Xibe ethnic group and Han populations from different regions.

Discrimination of Genetic Biomarkers of Disease through Machine-Learning-Based Hypothesis Testing of Direct SERS Spectra of DNA and RNA.

Cancer is globally a leading cause of death that would benefit from diagnostic approaches detecting it in its early stages. However, despite much research and investment, cancer early diagnosis is still underdeveloped. Owing to its high sensitivity, surface-enhanced Raman spectroscopy (SERS)-based detection of biomarkers has attracted growing interest in this area. Oligonucleotides are an important type of genetic biomarkers as their alterations can be linked to the disease prior to symptom onset. We propose a machine-learning (ML)-enabled framework to analyze complex direct SERS spectra of short, single-stranded DNA and RNA targets to identify relevant mutations occurring in genetic biomarkers, which are key disease indicators. First, by employing ad hoc-synthesized colloidal silver nanoparticles as SERS substrates, we analyze single-base mutations in ssDNA and RNA sequences using a direct SERS-sensing approach. Then, an ML-based hypothesis test is proposed to identify these changes and differentiate the mutated sequences from the corresponding native ones. Rooted in "functional data analysis," this ML approach fully leverages the rich information and dependencies within SERS spectral data for improved modeling and detection capability. Tested on a large set of DNA and RNA SERS data, including from miR-21 (a known cancer miRNA biomarker), our approach is shown to accurately differentiate SERS spectra obtained from different oligonucleotides, outperforming various data-driven methods across several performance metrics, including accuracy, sensitivity, specificity, and F1-scores. Hence, this work represents a step forward in the development of the combined use of SERS and ML as effective methods for disease diagnosis with real applicability in the clinic.