Shotgun metagenomics, from sampling to analysis

Abstract

Diverse microbial communities of bacteria, archaea, viruses and single-celled eukaryotes have crucial roles in the environment and in human health. However, microbes are frequently difficult to culture in the laboratory, which can confound cataloging of members and understanding of how communities function. High-throughput sequencing technologies and a suite of computational pipelines have been combined into shotgun metagenomics methods that have transformed microbiology. Still, computational approaches to overcome the challenges that affect both assembly-based and mapping-based metagenomic profiling, particularly of high-complexity samples or environments containing organisms with limited similarity to sequenced genomes, are needed. Understanding the functions and characterizing specific strains of these communities offers biotechnological promise in therapeutic discovery and innovative ways to synthesize products using microbial factories and can pinpoint the contributions of microorganisms to planetary, animal and human health.

Bidirectional transfer of Anelloviridae lineages between graft and host during lung transplantation

“…14,41 Thus, while the specific immune mechanisms responsible for regulating Anelloviridae in vivo are unknown, it has been proposed that monitoring levels in blood might serve as a "functional" measure of immune activity to manage organ transplant immunosuppression. Individual recipients where there was significant detection of these perioperative lineages in either BAL (A) or serum (B) are indicated by the asterisk (P < .05; Wilcoxon Rank Sum Test) 25,54 Illuminating this viral dark matter may reveal uncharacterized viruses that may play a role in lung health and disease. In addition, a novel observation here is that Anelloviridae within the lung allograft are also relatively de- A value of 1 is highly uneven coverage suggesting lack of detection while 0 is even coverage across the genome suggesting highly confident detection.…”

Functional variation in the gut microbiome of wild Drosophila populations

“…Sequence variation in the 16S rRNA gene is insufficient to represent functionally important strain variation caused by horizontal acquisition and deletion of genes in closely related bacteria (Gordienko, Kazanov & Gelfand, ; Jaspers & Overmann, ; Poretsky, Rodriguez, Luo, Tsementzi & Konstantinidis, ; Roux, Enault, Bronner & Debroas, ). The metagenome quantifies the genetic capacity for function, and some functional traits may remain undetected because of inadequate annotation (Quince, Walker, Simpson, Loman & Segata, ) or be inflated artificially by genes that are not expressed or are localized in metabolically inactive or recently killed microbial cells (Aguiar‐Pulido et al., ; Franzosa et al., ). Finally, the host gene expression profile is not a perfectly accurate representation of the conditions and resources encountered by the microorganisms in the gut because some genes are expressed in regions of the gut that are poorly colonized by microorganisms or are regulated post‐translationally.…”

Molecular genetic testing methodologies in hematopoietic diseases: current and future methods

“…A major pitfall of this approach is the inability to detect viruses, and information generated is restricted to 16SrDNA region. In the second approach (also called shotgun metagenomics), DNA is extracted from samples, whole genome libraries are prepared, and the samples are sequenced using NGS platform . Human DNA reads are filtered out, and the remaining reads are used to profile microbes either after their genome assembly (assembled de novo or assembled by mapping to microbial reference genomes) or by using assembly‐free metagenomic profiling approaches .…”