Hackflex library preparation enables low-cost metagenomic profiling.

Shotgun metagenomic sequencing provides valuable insights into microbial communities, but the high cost of library preparation with standard kits and protocols is a barrier for many. New methods such as Hackflex use diluted commercially available reagents to greatly reduce library preparation costs. However, these methods have not been systematically validated for metagenomic sequencing. Here, we evaluate Hackflex performance by sequencing metagenomic libraries from known mock communities as well as mouse fecal samples prepared by Hackflex, Illumina DNA Prep, and Illumina TruSeq methods. Hackflex successfully recovered all members of the Zymo mock community, performing best for samples with DNA concentrations <1 ng/uL. Furthermore, Hackflex was able to delineate microbiota of individual inbred mice from the same breeding stock at the same mouse facility, and statistical modeling indicated that mouse ID explained a greater fraction of the variance in metagenomic composition than did library preparation method. These results show that Hackflex is suitable for generating inventories of bacterial communities through metagenomic sequencing.

Hackflex library preparation enables low-cost metagenomic profiling.

Shotgun metagenomic sequencing provides valuable insights into microbial communities, but the high cost of library preparation with standard kits and protocols is a barrier for many. New methods such as Hackflex use diluted commercially available reagents to greatly reduce library preparation costs. However, these methods have not been systematically validated for metagenomic sequencing. Here, we evaluate Hackflex performance by sequencing metagenomic libraries from known mock communities as well as mouse fecal samples prepared by Hackflex, Illumina DNA Prep, and Illumina TruSeq methods. Hackflex successfully recovered all members of the Zymo mock community, performing best for samples with DNA concentrations <1 ng/µL. Furthermore, Hackflex was able to delineate microbiota of individual inbred mice from the same breeding stock at the same mouse facility, and statistical modeling indicated that mouse ID explained a greater fraction of the variance in metagenomic composition than did library preparation method. These results show that Hackflex is suitable for generating inventories of bacterial communities through metagenomic sequencing.

Home-site advantage for host species-specific gut microbiota.

Mammalian species harbor compositionally distinct gut microbial communities, but the mechanisms that maintain specificity of symbionts to host species remain unclear. Here, we show that natural selection within house mice (Mus musculus domesticus) drives deterministic assembly of the house-mouse gut microbiota from mixtures of native and non-native microbiotas. Competing microbiotas from wild-derived lines of house mice and other mouse species (Mus and Peromyscus spp.) within germ-free wild-type (WT) and Rag1-knockout (Rag1-/-) house mice revealed widespread fitness advantages for native gut bacteria. Native bacterial lineages significantly outcompeted non-native lineages in both WT and Rag1-/- mice, indicating home-site advantage for native microbiota independent of host adaptive immunity. However, a minority of native Bacteriodetes and Firmicutes favored by selection in WT hosts were not favored or disfavored in Rag1-/- hosts, indicating that Rag1 mediates fitness advantages of these strains. This study demonstrates home-site advantage for native gut bacteria, consistent with local adaptation of gut microbiota to their mammalian species.

Culture-enriched community profiling improves resolution of the vertebrate gut microbiota.

Vertebrates harbour gut microbial communities containing hundreds of bacterial species, most of which have never been cultivated or isolated in the laboratory. The lack of cultured representatives from vertebrate gut microbiotas limits the description and experimental interrogation of these communities. Here, we show that representatives from >50% of the bacterial genera detected by culture-independent sequencing in the gut microbiotas of fence lizards, house mice, chimpanzees, and humans were recovered in mixed cultures from frozen faecal samples plated on a panel of nine media under a single growth condition. In addition, culturing captured >100 rare bacterial genera overlooked by culture-independent sequencing, more than doubling the total number of bacterial sequence variants detected. Our approach recovered representatives from 23 previously uncultured candidate bacterial genera, 12 of which were not detected by culture-independent sequencing. Results identified strategies for both indiscriminate and selective culturing of the gut microbiota that were reproducible across vertebrate species. Isolation followed by whole-genome sequencing of 161 bacterial colonies from wild chimpanzees enabled the discovery of candidate novel species closely related to the opportunistic pathogens of humans Clostridium difficile and Hungatella hathewayi. This study establishes culturing methods that improve inventories and facilitate isolation of gut microbiota constituents from a wide diversity of vertebrate species.

The Impact of Heterogeneity on Single-Cell Sequencing.

The importance of diversity and cellular specialization is clear for many reasons, from population-level diversification, to improved resiliency to unforeseen stresses, to unique functions within metazoan organisms during development and differentiation. However, the level of cellular heterogeneity is just now becoming clear through the integration of genome-wide analyses and more cost effective Next Generation Sequencing (NGS). With easy access to single-cell NGS (scNGS), new opportunities exist to examine different levels of gene expression and somatic mutational heterogeneity, but these assays can generate yottabyte scale data. Here, we model the importance of heterogeneity for large-scale analysis of scNGS data, with a focus on the utilization in oncology and other diseases, providing a guide to aid in sample size and experimental design.

Epigenetic Modifications in Acute Myeloid Leukemia: Prognosis, Treatment, and Heterogeneity.

Leukemia, specifically acute myeloid leukemia (AML), is a common malignancy that can be differentiated into multiple subtypes based on leukemogenic history and etiology. Although genetic aberrations, particularly cytogenetic abnormalities and mutations in known oncogenes, play an integral role in AML development, epigenetic processes have been shown as a significant and sometimes independent dynamic in AML pathophysiology. Here, we summarize how tumors evolve and describe AML through an epigenetic lens, including discussions on recent discoveries that include prognostics from epialleles, changes in RNA function for hematopoietic stem cells and the epitranscriptome, and novel epigenetic treatment options. We further describe the limitations of treatment in the context of the high degree of heterogeneity that characterizes acute myeloid leukemia.