LICI Microbiome and Genetics Core

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

The Microbiome and Genetics core (MGC) of the Laboratory of Integrative Cancer Immunology (LICI) runs its microbiome facility in Building 37 of Bethesda with a team currently consisting of two research technicians, two bioinformaticians, two scientists and one postbac student. The microbiome has been implicated in numerous aspects of human health and the primary function of MGC is to provide a centralized facility that can meet the challenges of characterizing the role of the microbiota both in cancer and inflammatory processes as well as in general health. Microbiome characterizations are often case/control or longitudinal studies, sensitive to the methods and targets used to determine which microbes are present in which abundances, and which genes and pathways are enriched. Standardization in our methodology is crucial and was established early, using reliable and reproducible techniques to isolate and characterize nucleic acids of microbiota isolated from fecal sources. Robotic sample preparation platforms (Eppendorf 5073 and 5075) are used to maximize throughput and reproducibility, both for nucleic acid isolation and for barcoded library preparation. Quantification is accomplished using qPCR or spectroscopy. Following purification, barcoding and quantification, an Illumina MiSeq or NextSeq is used to sequence ribosomal gene amplicons or genomic shotgun libraries. The core works with a range of source materials and PIs to help effectively determine changes in microbial representation between experimental samples. The established process repertoire of the core consists of four distinct experimental characterizations. Ribosomal amplicon sequencing (16S for bacteria or ITS for fungi) is widely used to determine the identity of the microbes present in samples. Shotgun metagenomics and shotgun transcriptomics are becoming increasingly popular due to cost effectiveness and the detailed information on the genomic potential and real transcriptional activities of microbes present. Finally whole genome sequencing of microbial isolates is routinely undertaken. DNA and/or RNA has been extracted from source organisms such as human, mouse, macaque and drosophila and from source materials as varied as fecal pellets, anal and vaginal swabs, intestinal tissue and saliva. While amplicon sequencing enables identification of the taxa present, potential metabolic pathway changes induced by changes in gene content and composition of the microbiota can be tested with shotgun approaches. For genomic approaches, the same DNA isolation process is used and as little as 1ng of DNA is subjected to breakage and library preparation by transposon driven 'tagmentation'. Whole genome sequencing from isolates is done on the Illumina Miseq platform and shotgun metagenomes of the microbiota are run on the higher capacity Nexseq in the core or NovaSeq platforms elsewhere. The Nanopore MinION platform with lower throughput but longer reads is also being utilized to complement the deeper sequencing of Illumina platforms. In the past year several thousand samples have been processed for more than 20 projects from inside LICI and NCI as well as for collaborators from other NIH institutes and more than 0.5Tb of sequenced base pairs of data generated and analyzed. The MGC has incorporated experimental determination of absolute bacterial biomass estimates into projects focused on low biomass, utilizing known amounts of spike-in targets and both droplet digital PCR (ddPCR) alongside quantitative PCR methods. ddPCR works with limiting dilutions of DNA preparations containing single molecules, thus enabling minimization of contaminating host DNA. This capability can now be offered for low biomass experiments, enabling actual bacterial counts per unit of host tissue to be established. The MGC has continued to play a central part in characterizing the influence of microbiota in determining outcomes to immune checkpoint inhibitor therapy in melanoma as well as efficacy of fecal transplantation in cancer patients. In addition, the MGC has helped demonstrate enrichment of specific gram-negative genera is associated with immune activation and improved outcomes in therapy with immune checkpoint inhibition therapies. The influence of microbiota in modulating effects of combination therapies including CpG-containing neoadjuvents has also been a target of MGC's investigations over the past year, as has the role of exercise in favorably altering the metabolite output by microbiota during cancer immunotherapy leading to improved cytotoxic CD8 T-cell efficiency. In our computational work, developments over the past year include upgrading our sequencing data transfer process, to obtain a ~90% reduction in transfer completion time - an achievement that was featured in a FNLCR Sharepoint article. A redesigned version of JAMS, the MGC's package for producing genomic assembly and analysis starting from initial short reads through to assembled genomes, was publicly released on Github. The redesign incorporates methods that provide marked improvements in the percentage of assembly completion from microbiome data by providing additional analyses of unassigned contigs, helping to identify the taxa to which they belong. Ongoing support in genetics to research into immune receptors such as HLA and KIR continues, and the availability of health data from very large cohort studies has enabled more sensitive tracking of associations between immune markers and chronic disease. Members of the MGC have been co-authors on five publications over the past year with several papers in high-impact journals, including papers published in Cell also in Cancer Cell and in Immunity.