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Vaginal microbial flora analysis by next generation sequencing and microarrays; can microbes indicate vaginal origin in a forensic context?

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Abstract

Forensic analysis of biological traces generally encompasses the investigation of both the person who contributed to the trace and the body site(s) from which the trace originates. For instance, for sexual assault cases, it can be beneficial to distinguish vaginal samples from skin or saliva samples. In this study, we explored the use of microbial flora to indicate vaginal origin. First, we explored the vaginal microbiome for a large set of clinical vaginal samples (n = 240) by next generation sequencing (n = 338,184 sequence reads) and found 1,619 different sequences. Next, we selected 389 candidate probes targeting genera or species and designed a microarray, with which we analysed a diverse set of samples; 43 DNA extracts from vaginal samples and 25 DNA extracts from samples from other body sites, including sites in close proximity of or in contact with the vagina. Finally, we used the microarray results and next generation sequencing dataset to assess the potential for a future approach that uses microbial markers to indicate vaginal origin. Since no candidate genera/species were found to positively identify all vaginal DNA extracts on their own, while excluding all non-vaginal DNA extracts, we deduce that a reliable statement about the cellular origin of a biological trace should be based on the detection of multiple species within various genera. Microarray analysis of a sample will then render a microbial flora pattern that is probably best analysed in a probabilistic approach.

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Acknowledgements

We thank Frank Schuren, Anita Ouwens and Michel Ossendrijver (TNO Quality of Life, The Netherlands) for the development of the (vaginal) microbial flora microarray and Hans Korporaal for retrieving the 240 clinical samples from the archives of the Leiden Cytology and Pathology Laboratory (The Netherlands). We are thankful to the donors who provided the various types of samples and we thank Rolla Voorhamme for critically reading the manuscript.

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Authors and Affiliations

  1. Netherlands Forensic Institute, Laan van Ypenburg 6, 2497GB, The Hague, The Netherlands

    Corina C. G. Benschop, Frederike C. A. Quaak, Titia Sijen & Irene Kuiper

  2. Leiden Cytology and Pathology Laboratory, P.O. Box 16084, 2301 GB, Leiden, The Netherlands

    Mathilde E. Boon

Authors
  1. Corina C. G. Benschop
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  2. Frederike C. A. Quaak
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  3. Mathilde E. Boon
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  4. Titia Sijen
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  5. Irene Kuiper
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Corresponding author

Correspondence to Titia Sijen.

Additional information

Both C. C. G. Benschop and F. Quaak contributed equally. M. Boon is the pathologist responsible for the morphologic evaluation of the cervical brush samples.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1

Overview of age pools with number of samples and reads per pool. Reads are grouped in genera represented by different colors. White bars represent Lactobacillus, green bars represent Gardnerella, other colours/genera are not specified (PDF 42 kb)

Supplementary Fig. 2

Distribution of next generation sequencing reads at genus level. Sequencing reads are based on 240 clinical vaginal samples and were assigned to genera using RDP. The percentages of sequence reads per genus are also incorporated in Supplementary Table 2 (PDF 148 kb)

Supplementary Fig. 3

Distribution of next generation sequencing reads assigned to species within the genus Lactobacillus. Sequencing reads are based on 240 clinical vaginal samples. A full overview of reads within the genus Lactobacillus is incorporated in Supplementary Table 3 (PDF 146 kb)

Supplementary Fig. 4

Total number of probes (A) and genera (B) detected using microarray analysis of NF and SF extracts obtained from the same swab (n=9). Genera assigned as Gram positive, Gram negative and Gram variable are shown as black, grey and white bars, respectively. (DOC 30 kb)

Supplementary Table1

Primer sequences used in various experiments (PDF 6 kb)

Supplementary Table 2

Percentage of next generation sequence reads on genus level based on 240 clinical cervical brush samples. Total number of sequence reads: 338,184 (PDF 8 kb)

Supplementary Table 3

Percentage of next generation sequence reads for Lactobacillus species based on 240 clinical cervical brush samples. Total number of Lactobacillus sequence reads: 199,433 (PDF 6 kb)

Supplementary Table 4

Percentage of samples per body site which were positive for a specific genus or family that was either detected in vaginal samples (vaginal +) or not detected in vaginal samples (vaginal-). A sample is regarded positive if at least one probe per genus or family is detected. For positive samples the number per total number of detected probes is shown for each genus (# of probes) (PDF 84 kb)

Supplementary Table 5

Distribution of 21 probes exclusively detected in vaginal DNA extracts. (A) Number (%) of vaginal DNA extracts detected per probe. (B) Number of probes detected per vaginal DNA extract (PDF 5 kb)

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Benschop, C.C.G., Quaak, F.C.A., Boon, M.E. et al. Vaginal microbial flora analysis by next generation sequencing and microarrays; can microbes indicate vaginal origin in a forensic context?. Int J Legal Med 126, 303–310 (2012). https://doi.org/10.1007/s00414-011-0660-8

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  • DOI: https://doi.org/10.1007/s00414-011-0660-8

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