Optimization of DNA extraction and sampling methods for successful forensic microbiome analyses of the skin and saliva.
Author(s): Yu KM, Lee AM, Cho HS, Lee JW, Lim SK
Publication: Int J Legal Med, 2023, Vol. 137, Page 63-77
PubMed ID: 36416962 PubMed Review Paper? No
Purpose of Paper
This paper compared the microbiome of case-matched saliva and scalp specimens collected using two different types of cotton and nylon swabs and extracted using four different kits.
Conclusion of Paper
Saliva specimens had significantly higher alpha diversity than scalp specimens. In principal coordinate analysis (PCoA) based on Bray-Curtis distances, cotton swab specimens from saliva clustered separately from scalp specimens. Microbiome taxa from saliva and scalp specimens differed greatly on the family level. Saliva specimens had more species overall, but scalp specimens had much greater dispersion.
Concentrations of total and human DNA from scalp and saliva specimens were dependent on the DNA extraction method. For scalp specimens, the highest total yields were obtained with QIAamp Mini and PrepFiler Kits (3.803 ng/μL and 3.884 ng/μL, respectively), but the highest concentration of human DNA was obtained with the Maxwell Kit (0.090 ng/μL). For saliva specimens, significantly higher total DNA yields were obtained with the Maxwell Kit (23.767 ng/μL) than any of the other three kits, but the highest concentration of human DNA was obtained with the PrepFiler Kit (18.976 ng/μL versus 10.126 ng/μL, 0.657 ng/μL and 0.163 ng/μL using the Maxwell, QIAamp Micro and QIAamp Mini Kits, respectively). The DNA extraction kit affected the relative abundance of species at the genus level, but PCoA based on Bray-Curtis distances did not cluster specimens by extraction method. The authors report that the Maxwell and PrepFiler Kits were better for detecting rare genera.
When cotton and nylon collection swabs were compared, scalp specimens collected with nylon swabs had higher total and human DNA yields than cotton swabs. In saliva specimens, the highest yields of total DNA were obtained using the COPAN nylon swabs (2.337 ng/μL) followed by the Puritan cotton swabs (1.989 ng/μL); however, the human DNA yields from saliva stains were slightly higher with nylon (1.959 ng/μL for COPAN and 1.625 ng/μL for NobleBIo) than cotton swabs (1.596 ng/μL for Puritan and 1.476 ng/μL for Poongsung). Bacterial DNA band intensities were comparable among swab types. Alpha diversity was slightly higher for specimens collected with cotton swabs, but nylon swab specimens had a higher number of low abundance genera.
Studies
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Study Purpose
This study compared the microbiome of case-matched saliva and scalp specimens collected using two different types of cotton and nylon swabs and extracted using four different kits. Skin and saliva were collected from two healthy women and one healthy man. Participants refrained from showering for 8 h before collecting skin specimens by rubbing a wet swab on the scalp for 30 s; swab specimens were then air-dried overnight at room temperature and stored frozen at -70°C until DNA extraction. Saliva specimens were collected > 2 h after eating by an unspecified method. For DNA extraction method comparisons, DNA was extracted from cotton swab scalp specimens and saliva using the QIAamp DNA Mini Kit, the QIAamp DNA Micro Kit, the Maxwell FSC DNA IQTM Casework Kit and the PrepFiler Forensic DNA Extraction Kit. To investigate the effects of swab type, a saliva smear was created, and scalp and saliva specimens were collected by rubbing wet Poongsang and Puritan cotton swabs and NobleBio and COPAN Nylon swabs on the scalp and saliva stains; DNA from different swab types was extracted using the MAXwell FSC DNA IQ Kit for comparisons. DNA was quantified using the Tecan F200 Kit and a Quantus Fluorometer and by real-time PCR using the Attoplex Human Genomic DNA Quantification Kit. The V3-V4 region of 16S rRNA genes were amplified and sequenced with an Illumina NovaSeq6000 instrument.
Summary of Findings:
Saliva specimens had higher alpha diversity than scalp specimens using both measures of richness alone (Observed amplicon sequence variants P= 9.2557e-07, Chao1 P=1.6456e-06 and Abundance-based Coverage Estimator (ACE) P= 2.0581e-06) and measures of richness and evenness (Shannon P= 1.6104e-05, Simpson P= 1.5086e-05 and Fisher P= 1.8533e-06). Importantly, saliva and scalp specimens differed greatly on the family level; while saliva specimens had more species, they were more similar to each other, with much greater dispersion noted for the scalp specimens. In principal coordinate analysis (PCoA) based on Bray-Curtis distances, cotton swab specimens from saliva clustered separately from scalp specimens.
QIAamp Mini and PrepFiler Kits yielded the highest total DNA concentrations (3.803 ng/μL and 3.884 ng/μL, respectively) from scalp specimens, but differences among the four extraction kits were not statistically significant. Interestingly, the specimen with the highest total DNA concentration was not the specimen with the highest concentration of human DNA. The highest concentration of human DNA was obtained using the Maxwell Kit (0.090 ng/μL), followed by QIAamo Mini (0.065 ng/μL), PrepFiler (0.064 ng/μL) and QIAami Micro Kits (0.006 ng/μL), respectively. For saliva specimens, significantly higher total DNA yields were obtained with the Maxwell Kit (23.767 ng/μL) than any of the other three kits (5.796 ng/μL for PrepFiler, 3.894 ng/μL for QIAamp Mini and 3.694 ng/μL for QIAamp Micro Kit), but the highest concentration of human DNA was obtained using the PrepFiler Kit (18.976 ng/μL versus 10.126 ng/μL, 0.657 ng/μL and 0.163 ng/μL using the Maxwell, QIAamp Micro and QIAamp Mini Kits, respectively). DNA extraction kit affected the relative abundance of species at the genus level, but PCoA based on Bray-Curtis distances did not cluster specimens by extraction method. The authors reported that the Maxwell and PrepFiler Kits were better for the detection of rare genera.
Yields of total and human DNA from scalp specimens were higher when nylon swabs were used for collection (0.428 ng/μL and 0.308 ng/μL, respectively for NobleBio and 0.370 ng/μL and 0.198 ng/μL, respectively for COPAN) compared to cotton swabs (0.227 ng/μL and 0.061 ng/μL, respectively for Poonsung swabs and 0.209 ng/μL and 0.057 ng/μL, respectively for Puritan swabs). The highest yields of total DNA from saliva stains were obtained with COPAN nylon swabs (2.337 ng/μL), followed by Puritan cotton swabs (1.989 ng/μL); however, yields of human DNA from saliva stains were slightly higher when using nylon swabs (1.959 ng/μL for COPAN and 1.625 ng/μL for NobleBIo) rather than cotton swabs (1.596 ng/μL for Puritan and 1.476 ng/μL for Poongsung). Bacterial DNA band intensities were comparable among swab types. Alpha diversity was slightly higher for specimens collected with cotton swabs (P-values not given), but the nylon swabs had a higher number of low-abundance genera.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry DNA Next generation sequencing DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Scalp
Saliva
Analyte Extraction and Purification Analyte isolation method QIAamp DNA Mini Kit
QIAamp DNA Micro Kit
Maxwell FSC DNA IQTM Casework Kit
PrepFiler Forensic DNA Extraction Kit
Biospecimen Acquisition Method of cell acquisition Poongsang cotton swab
Puritan cotton swab
NobleBio nylon swab
COPAN nylon swab