Determining Informative Microbial Single Nucleotide Polymorphisms for Human Identification.

The skin microbiome is a highly abundant and relatively stable source of DNA that may be utilized for human identification (HID). In this study, a set of single nucleotide polymorphisms (SNPs) with a high mean estimated Wright's fixation index (FST) (>0.1) and widespread abundance (found in ≥75% of samples compared) were selected from a diverse set of markers in the hidSkinPlex panel. The least absolute shrinkage and selection operator (LASSO) was used in a novel machine learning framework to generate a SNP panel and predict the human host from skin microbiome samples collected from the hand, manubrium, and foot. The framework was devised to emulate a new unknown person introduced to the algorithm and to match samples from that person against a population database. Unknown samples were classified with 96% accuracy (Matthews correlation coefficient [MCC], 0.954) in the test (n = 225 samples) data set. A final panel of informative SNPs was determined for HID (hidSkinPlex+) using all 51 individuals sampled at three body sites in triplicate. The hidSkinPlex+ panel comprises 365 SNPs and yielded prediction accuracy for the correct host of 95% (MCC = 0.949). The accuracy of the hidSkinPlex+ panel may be somewhat overestimated due to using 26 individuals from the training data set for the selection of the final panel. However, this accuracy still provides an indication of performance when tested on new samples. IMPORTANCE One of the fundamental goals in forensic genetics is to identify the source of biological evidence. Methods for detecting human DNA have advanced and can be quite sensitive, but not all DNA samples are amenable to current methods. However, the human skin microbiome is a source of DNA with high copy numbers, and it has the potential for high discriminatory power. The hidSkinPlex panel has been used for HID; however, some aspects of it could be improved. Missing information is ambiguous, as it is unclear if marker drop-out is a by-product of a low-template sample or if the reasons for not observing a marker are biological. Such ambiguity may confound methods for HID, and as such, an improved marker set (hidSkinPlex+) was designed that is considerably smaller and more robust to drop-out (365 SNPs contained in 135 markers) yet still can be used to accurately predict the human host.

Population Informative Markers Selected Using Wright's Fixation Index and Machine Learning Improves Human Identification Using the Skin Microbiome.

Microbial DNA, shed from human skin, can be distinctive to its host and, thus, help individualize donors of forensic biological evidence. Previous studies have utilized single-locus microbial DNA markers (e.g., 16S rRNA) to assess the presence/absence of personal microbiota to profile human hosts. However, since the taxonomic composition of the microbiome is in constant fluctuation, this approach may not be sufficiently robust for human identification (HID). Multimarker approaches may be more powerful. Additionally, genetic differentiation, rather than taxonomic distinction, may be more individualizing. To this end, the nondominant hands of 51 individuals were sampled in triplicate (n = 153). They were analyzed for markers in the hidSkinPlex, a multiplex panel comprising candidate markers for skin microbiome profiling. Single-nucleotide polymorphisms (SNPs) with the highest Wright's fixation index (FST) estimates were then selected for predicting donor identity using a support vector machine (SVM) learning model. FST is an estimate of the genetic differences within and between populations. Three different SNP selection criteria were employed: SNPs with the highest-ranking FST estimates (i) common between any two samples regardless of markers present (termed overall); (ii) each marker common between samples (termed per marker); and (iii) common to all samples used to train the SVM algorithm for HID (termed selected). The SNPs chosen based on criteria for overall, per marker, and selected methods resulted in an accuracy of 92.00%, 94.77%, and 88.00%, respectively. The results support that estimates of FST, combined with SVM, can notably improve forensic HID via skin microbiome profiling. IMPORTANCE There is a need for additional genetic information to help identify the source of biological evidence found at a crime scene. The human skin microbiome is a potentially abundant source of DNA that can enable the identification of a donor of biological evidence. With microbial profiling for human identification, there will be an additional source of DNA to identify individuals as well as to exclude individuals wrongly associated with biological evidence, thereby improving the utility of forensic DNA profiling to support criminal investigations.

Copan microFLOQ® Direct Swab collection of bloodstains, saliva, and semen on cotton cloth.

The microFLOQ® Direct Swab was tested by sampling diluted blood, semen, and saliva stains deposited on cotton cloth. DNA typing was performed using the PowerPlex® Fusion 6C System by direct PCR or a modified direct PCR. Direct PCR of swabs sampled the center of a stain, compared to their respective edge samplings, and had higher profile completeness and total relative fluorescent units (RFU) for all dilutions of blood and semen stains tested. The modified direct PCR used template DNA eluted from the swab head using the Casework Direct Kit, Custom and washes either contained 1-thioglycerol (TG) additive or no TG. Modified direct PCR had mixed results for blood, saliva, and semen stains, with semen stains showing significant differences in profile completeness (5% and 1%) and total RFU (neat, 5% and 1%) with the addition of TG to the Casework Direct Reagent. No significant difference was seen in any dilution of blood or saliva stains processed with the modified direct PCR, but profile completeness and total RFU were improved overall compared to stains swabbed with cotton swabs or 4N6FLOQSwabs™. This study supports the hypothesis that the microFLOQ® Direct Swab is able to collect minute amounts of DNA from cotton cloth and may be considered as an alternate pre-screening methodology in forensic biology casework.