Dataset Information

Analysis of red autofluorescence (650-670nm) in epidermal cell populations and its potential for distinguishing contributors to 'touch' biological samples.

ABSTRACT: Interpretation of touch DNA mixtures poses a significant challenge for forensic caseworking laboratories. Front end techniques that facilitate separation of contributor cell populations before DNA extraction are a way to circumvent this problem. The goal of this study was to survey intrinsic fluorescence of epidermal cells collected from touch surfaces and investigate whether this property could potentially be used to discriminate between contributor cell populations in a biological mixture. Analysis of red autofluorescence (650-670nm) showed that some contributors could be distinguished on this basis. Variation was also observed between autofluorescence profiles of epidermal cell populations from a single contributor sampled on different days. This dataset suggests that red autofluorescence may be a useful marker for identifying distinct cell populations in some mixtures. Future efforts should continue to investigate the extrinsic or intrinsic factors contributing to this signature, and to identify additional biomarkers that could complement this system.

SUBMITTER: Stanciu CE

PROVIDER: S-EPMC5130068 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Analysis of red autofluorescence (650-670nm) in epidermal cell populations and its potential for distinguishing contributors to 'touch' biological samples.

Stanciu Cristina E CE Philpott M Katherine MK Bustamante Eduardo E EE Kwon Ye Jin YJ Ehrhardt Christopher J CJ

F1000Research 20160216

Interpretation of touch DNA mixtures poses a significant challenge for forensic caseworking laboratories. Front end techniques that facilitate separation of contributor cell populations before DNA extraction are a way to circumvent this problem. The goal of this study was to survey intrinsic fluorescence of epidermal cells collected from touch surfaces and investigate whether this property could potentially be used to discriminate between contributor cell populations in a biological mixture. A ...[more]

PMID: 27990254

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Project description:The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650-670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor's contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650-670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures. Graphical Abstract Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.

Project description:Lactic acid bacteria (LAB) are important for human health. However, the relative abundance of LAB in complex samples, such as fecal samples, is low and their presence and diversity (at the species level) is understudied. Therefore, we designed LAB-specific primer pairs based on 16S rRNA gene consensus sequences from 443 species of LAB from seven genera. The LAB strains selected were genetically similar and known to play a role in human health. Prior to primer design, we obtained consistent sequences for the primer-binding sites by comparing the 16S rRNA gene sequences, manually identifying single-stranded primers and modifying these primers using degenerate bases. We assembled primer pairs with product sizes of >400 bp. Optimal LAB-specific primers were screened using three methods: PCR amplification, agarose gel electrophoresis and single-molecule real-time (SMRT) sequencing analysis. During the SMRT analysis procedure, we focused on sequence reads and diversity at the species level of target LAB in three fecal samples, using the universal bacterium primer 27f/1492r as a reference control. We created a phylogenetic tree to confirm the ability of the best candidate primer pair to differentiate amongst species. The results revealed that LAB-specific primer L5, with a product size of 750 bp, could generate 3222, 2552, and 3405 sequence reads from fecal Samples 1, 2, and 3. This represented 14, 13 and 10% of all target LAB sequence reads, respectively, compared with 2, 0.8, and 0.8% using the 27f/1492r primer. In addition, L5 detected LAB that were in low abundance and could not be detected using the 27f/1492r primer. The phylogenetic tree based on the alignments between the forward and reverse primer of L5 showed that species within the seven target LAB genera could be distinguished from each other, confirming L5 is a powerful tool for inferring phylogenetic relationships amongst LAB species. In conclusion, L5 is a LAB-specific primer that can be used for high-throughput sequencing and identification of taxa to the species level, especially in complex samples with relatively low LAB content. This enables further research on LAB population diversity in complex ecosystem, and on relationships between LAB and their hosts.

Dataset Information

Analysis of red autofluorescence (650-670nm) in epidermal cell populations and its potential for distinguishing contributors to 'touch' biological samples.

Publications

Analysis of red autofluorescence (650-670nm) in epidermal cell populations and its potential for distinguishing contributors to 'touch' biological samples.

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