Microbial enrichment and storage for metagenomics of vaginal, skin, and saliva samples

Abstract

Summary Few validated protocols are available for large-scale collection, storage, and analysis of microbiome samples from the vagina, skin, and mouth. To prepare for a large-scale study on the female microbiome by remote self-sampling, we investigated the impact of sample collection, storage, and host DNA depletion on microbiome profiling. Vaginal, skin, and saliva samples were analyzed using 16S rRNA gene amplicon and metagenomic shotgun sequencing, and qPCR. Of the two tested storage buffers, the eNAT buffer could keep the microbial composition stable during various conditions. All three tested host DNA-depletion approaches showed a bias against Gram-negative taxa. However, using the HostZERO Microbial DNA and QIAamp DNA Microbiome kits, samples still clustered according to body site and not by depletion approach. Therefore, our study showed the effectiveness of these methods in depleting host DNA. Yet, a suitable approach is recommended for each habitat studied based on microbial composition.

DNA Enrichment Methods for Microbial Symbionts in Marine Bivalves

“…Moreover, we found that the DNA extracted with HostZERO Microbial DNA kit had lower relative abundances of most gram-negative taxa in comparison to PowerSoil DNA extraction kit. This bias has also been observed in the saliva microbial community [41]. It might be that the relative abundance of gram-negative bacteria was "diluted" by the DNA of gram-positive bacteria.…”

“…These results suggest that the high DNA yields acquired using methods PS, PC, and PF are probably attributed to the involvement of host (gill tissue) DNA. The HostZERO Microbial DNA kit has also been reported to have had good host depletion efficiency in coral and clinical samples [24,41,43]. That means selective lysis of eukaryotic host cells followed by removal of exposed DNA could be an effective method of reducing host-aligned sequencing reads, and it is also suitable for enriching microbial DNA from bivalve gill tissues.…”

“…Employing QIAamp DNA Microbiome kit acquired higher bacterial DNA yields from oyster tissue, while the microbial diversity was reduced [23]. Though the HostZERO Microbial DNA kit could effectively deplete host DNA in clinical samples [41], food [42], and coral [43], it has not been applied in host DNA depletion for bivalve samples. In addition, Ahannach et al, 2021 found that different microbial DNA enrichment methods can introduce some biases into the microbial diversity profile and cause potential taxonomic composition differences in skin and saliva samples [41].…”

“…Though the HostZERO Microbial DNA kit could effectively deplete host DNA in clinical samples [41], food [42], and coral [43], it has not been applied in host DNA depletion for bivalve samples. In addition, Ahannach et al, 2021 found that different microbial DNA enrichment methods can introduce some biases into the microbial diversity profile and cause potential taxonomic composition differences in skin and saliva samples [41]. To date, the efficiency of these different methods for depleting host DNA from bivalve tissues and the bias introduced by these methods to the composition of these samples have rarely been evaluated [23].…”

Spontaneous Riboflavin-Overproducing Limosilactobacillus reuteri for Biofortification of Fermented Foods

“…The merged and denoised reads (amplicon sequence variants or ASVs) were taxonomically annotated from the phylum to the genus level with the assignTaxonomy function of DADA2 using the EzBioCloud reference 16S rRNA database ( 39 ). All data handling and visualization was performed in R version 3.4.4 ( 40 ) using the tidyverse set of packages and the in-house package tidyamplicons 6 ( 41 ).…”