Decontamination strategy for detection of cfmDNA in humans

Madhavi Bhatia, NIPER Guwahati.

The gut flora of humans acts as a major factor that influences the health of an individual. The microbiome may affect the disease progression and response to immunotherapy. So, the gut microbiome can act as a cancer biomarker. For analyzing this high throughput 16S-rRNA-gene-based approach is utilized to detect a genuine cell-free microbial DNA(cfmDNA) signal between healthy and cancer patients.

A cohort study was conducted by using 69 stage IV melanoma patients, DNA was extracted from their plasma, saliva and stool samples. Along with this DNA was extracted from negative controls. Concentration levels of microbial DNA in the sample and negative control was determined using 16S-rRNA-gene droplet digital PCR (ddPCR) assay. The microbial DNA concentration in plasma was much lower than the concentration measured in stool and saliva. However, the overall microbial DNA concentration in plasma was greater than its corresponding negative control and the difference in concentration was statistically significant. It was also found that microbial DNA concentration levels of plasma negative control samples were significantly higher than the negative control samples of stool and saliva. Thus, it suggests that plasma DNA extraction kit reagents had a higher amount of contaminated has compared to stool and saliva DNA extraction kits used. (Fig 1)

Microbial community analysis of cell-free microbial DNA–

Phylogenetic resolution of the samples was done by sequencing the reads obtained by the samples of plasma, saliva and stool which generated amplicon sequence variants(ASV) using the Illumina MiSeq platform. Upon taxonomic profiling of samples, it was observed that microbiome composition differed between sample types, these differences increased with taxonomic level depth. The phylum-level taxonomic profiles of stool and saliva samples significantly differed from their DNA negative control. However, there was a higher similarity in the taxonomic profiling of plasma sample and their DNA negative control. This indicates that the plasma microbiome is highly susceptible to contaminated DNA.

In Silico decontamination– 

In Silico decontamination is used as a strategy to specifically remove contaminant DNA from our datasets and to obtain genuine ASVs. Three bioinformatics decontamination criteria which ASV must meet in order to be high confidence plasma ASV- a. No significant differential abundance due to technical variables that cause sequencing variation. b. A high prevalence in plasma vs plasma DENC samples. c. ASVs present only in patient-matching samples to have a significant association in the detection of the ASV between replicates using inter-rater reliability kappa score>0.4. Most of these ASVs were classified into genera of bacteria commonly found in gut-Faecalibacterium, Bacteroids and Ruminococcus. Only one high confidence plasma ASV of the g genus Veillonella was found in the saliva sample. Thus, In Silico decontamination suggest that the gut microbiome may represent a significant source of cfmDNA.

Identification of cfmDNA in healthy individuals and cancer patients–

CfmDNA profile of healthy individuals and cancer patients was identified by applying the 16S RNA gene sequencing method and then analyzed. Taxonomic profiling exhibited a high similarity between plasma sample and negative control samples of plasma for both healthy individuals and melanoma patients. ASVs showed a significant difference between the microbiome compositions of cfmDNA from healthy individuals and their negative controls. Castellaniella genus was observed only in healthy individuals and was recently identified as a genuine cfmDNA member.

With the help of the 16S-rRNA gene sequencing approach, it was found that plasma from healthy individuals and cancer patients have a low yet detectable level of cfmDNA, which helps in the identification of potential differences in the cfmDNA between healthy and cancer patients. By performing whole metagenomics sequencing analysis of cfDNA from plasma along with contamination controls, distinct cancer-type specific cfmDNA signatures were identified. Thus, cfmDNA based diagnosis has a huge potential in cancer, when appropriate decontamination strategies are applied to remove the contamination by DNA.

Also read: Pancreatic Cancer Stem Cells & Tumorigenesis

Source-Zozaya-Valdés, E., Wong, S.Q., Raleigh, J. et al. Detection of cell-free microbial DNA using a contaminant-controlled analysis framework. Genome Biol22, 187 (2021). https://doi.org/10.1186/s13059-021-02401-3.

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