Tracing mother-infant transmission of bacteriophages by means of a novel analytical tool for shotgun metagenomic datasets: METAnnotatorX

Abstract

BackgroundDespite the relevance of viral populations, our knowledge of (bacterio) phage populations, i.e., the phageome, suffers from the absence of a “gold standard” protocol for viral DNA extraction with associated in silico sequence processing analyses. To overcome this apparent hiatus, we present here a comprehensive performance evaluation of various protocols and propose an optimized pipeline that covers DNA extraction, sequencing, and bioinformatic analysis of phageome data.ResultsFive widely used protocols for viral DNA extraction from fecal samples were tested for their performance in removal of non-viral DNA. Moreover, we developed a novel bioinformatic platform, METAnnotatorX, for metagenomic dataset analysis. This in silico tool facilitates a range of read- and assembly-based analyses, including taxonomic profiling using an iterative multi-database pipeline, classification of contigs at genus and species level, as well as functional characterizations of reads and assembled data. Performances of METAnnotatorX were assessed through investigation of seven mother-newborn pairs, leading to the identification of shared phage genotypes, of which two were genomically decoded and characterized.METAnnotatorX was furthermore employed to evaluate a protocol for the identification of contaminant non-viral DNA in sequenced datasets and was exploited to determine the amount of metagenomic data needed for robust evaluation of human adult-derived (fecal) phageomes.ConclusionsResults obtained in this study demonstrate that a comprehensive pipeline for analysis of phageomes will be pivotal for future explorations of the ecology of phages in the gut environment as well as for understanding their impact on the physiology and bacterial community kinetics as players of dysbiosis and homeostasis in the gut microbiota.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0527-z) contains supplementary material, which is available to authorized users.

Oral bacteriophages are maintained at high levels for months in individuals but infrequently transmitted between mothers and infants

“…In the single case of phage 20 where a very similar phage was present in a mother and baby from the same family, the strain was as different (1.75% nucleotide divergence) as many phage from unrelated subjects, suggesting that it was acquired from a different source. Other studies have shown transmission of phage from mothers to babies (52–54) so it is likely there was not a large enough sample size of phage and subjects to observe it here. Transmission does seem to be less frequent than maintenance within an individual.…”

Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach

“…3 ; Data Set S4 ). Of note, the discriminatory power of microbial ITS-based profiling is dependent on the availability of reference ITSs extracted from genomic sequences, similar to profiling methods based on shotgun metagenomics data ( 7 , 8 , 40 – 42 ). Therefore, the percentage of classified species is expected to increase due to ongoing genome decoding of novel bacterial species.…”

“…Moreover, bases were removed from the end of the reads unless the average quality score was >25, in a window of 5 bp. Quality-filtered data were used to further analysis with METAnnotatorX ( 8 ) and MetaPhlAn2 ( 42 ) for taxonomic profile reconstruction. Both software packages were used with default settings, and the RefSeq database obtained from NCBI in October 2019 was used for taxonomic classification using METAnnotatorX ( 8 ).…”

“…Currently, shotgun and long-read sequencing methods provide higher taxonomic resolution, although their higher cost represents an important limitation compared to the use of 16S rRNA-based microbial profiling approach ( 7 – 9 ). Moreover, the absence of a PCR amplification step may prevent retrieval of sufficient microbial DNA for analysis of host-associated, low-abundance bacterial communities due to interfering levels of eukaryotic DNA ( 7 ).…”

“…ITS sequences are characterized by higher variability than 16S rRNA genes, thus allowing (sub)species taxonomic resolution when employed for metagenomic profiling purposes ( 10 – 12 ). Remarkably, the ITS profiling method combines the lower cost and high sensitivity of a marker gene amplification approach with the resolution of shotgun metagenomics ( 7 , 8 ).…”