Next‐generation sequencing approach to epigenetic‐based tissue source attribution

Abstract

The ability to determine the tissue source of biological materials from evidence samples can be highly informative for interpreting forensic data. In this study, a previously published CE‐based method to probe locus‐specific DNA methylation was modified to accommodate detection using next‐generation sequencing (NGS) to perform tissue source attribution. DNA samples (1 ng) from each of four different tissue types were digested with the methylation sensitive restriction endonuclease Hha1 and PCR was used to amplify an optimized subset of ten methylated loci, including positive and negative control loci. The products were prepared as NGS libraries, pooled in a multiplex assay with sample‐specific barcodes, sequenced with an Illumina MiSeq, and analyzed using a k‐Nearest Neighbor algorithm. With this initial effort a concordance rate of 15/16 was demonstrated from samples of varying types: semen, saliva, skin epidermis, and blood. This method also was designed to be compatible with the workflows published to date for NGS of STRs. Thus, the methylation approach described here is highly accurate and upon further validation and testing may be potentially used in practice as a confirmatory test in conjunction with other NGS protocols used in forensic laboratories.

Alteration in the expression of MGMT and RUNX3 due to non-CpG promoter methylation and their correlation with different risk factors in esophageal cancer patients

“…MiSeq sequencing system-Illumina technology was used for NGS that provides the highest accuracy over other methylome analytical methods. 33,34 NGS provides the additional advantage of determining the non-CpG (CHG and CHH) methylation status along with CpG. MGMT mediated DNA repair is the simplest form of DNA repair which is the direct removal of the methyl adducts from altered gDNA.…”

Discovery of potential DNA methylation markers for forensic tissue identification using bisulphite pyrosequencing

“…Future experiments using semen stains of decreasing size (as measured in microlitres) would fully determine the assays’ sensitivity proposed in this study. Novel next‐generation sequencing (NGS), including various NGS platforms, has been increasingly used in the forensic field and recent studies have also investigated its applicability in tissue identification using DNA methylation markers . Applying NGS could significantly increase the sensitivity and also allow for analysis of an increased number of tissue‐specific markers included in a tissue‐ID assay.…”

Forensic body fluid identification: state of the art

“…New approaches combining epigenetic analysis and MPS for body fluid identification are appearing in the literature and may provide a fresh impetus for discovering stable markers that do not change with influences such as age and environment. 126…”