Heike Sichtig’s research while affiliated with U.S. Food and Drug Administration and other places

FDA proactively invests in tools to support innovation of emerging technologies, such as infectious disease next generation sequencing (ID-NGS). Here, we introduce FDA-ARGOS quality-controlled reference genomes as a public database for diagnostic purposes and demonstrate its utility on the example of two use cases. We provide quality control metrics for the FDA-ARGOS genomic database resource and outline the need for genome quality gap filling in the public domain. In the first use case, we show more accurate microbial identification of Enterococcus avium from metagenomic samples with FDA-ARGOS reference genomes compared to non-curated GenBank genomes. In the second use case, we demonstrate the utility of FDA-ARGOS reference genomes for Ebola virus target sequence comparison as part of a composite validation strategy for ID-NGS diagnostic tests. The use of FDA-ARGOS as an in silico target sequence comparator tool combined with representative clinical testing could reduce the burden for completing ID-NGS clinical trials.

The DNA base modification N6-methyladenine (m⁶A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m⁶A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.

Infectious disease next generation sequencing (ID-NGS) diagnostics are on the cusp of revolutionizing the clinical market. To facilitate this transition, FDA proactively invested in tools to support innovation of emerging technologies. FDA and collaborators established a publicly available database, FDA dAtabase for Regulatory-Grade micrObial Sequences (FDA-ARGOS), as a tool to fill reference database gaps with quality-controlled genomes. This manuscript discusses quality control metrics for the proposed FDA-ARGOS genomic resource and outlines the need for quality-controlled genome gap filling in the public domain. Here, we also present three case studies showcasing potential applications for FDA-ARGOS in infectious disease diagnostics, specifically: assay design, reference database and in silico sequence comparison in combination with representative microbial organism wet lab testing; a novel composite validation strategy for ID-NGS diagnostics. The use of FDA-ARGOS as an in silico comparator tool could reduce the burden for completing ID-NGS clinical trials. In addition, use cases identifying Enterococcus avium and Ebola virus (Zaire ebolavirus variant Makona) demonstrate the utility of FDA-ARGOS as a reference database for independent performance validation of new tests and for documenting how one would use this database as an in silico sequence target comparator tool for ID-NGS validation, respectively.

Staphylococcus aureus is a major human pathogen with well-characterized bacteriophage contributions to its virulence potential. Recently, we identified plasmidial and episomal prophages in S. aureus strains using an extra-chromosomal DNA (exDNA) isolation and sequencing approach, uncovering the plasmidial phage ϕBU01, which was found to encode important virulence determinants. Here, we expanded our extra-chromosomal sequencing of S. aureus, selecting 15 diverse clinical isolates with known chromosomal sequences for exDNA isolation and next-generation sequencing. We uncovered the presence of additional episomal prophages in 5 of 15 samples, but did not identify any plasmidial prophages. exDNA isolation was found to enrich for circular prophage elements, and qPCR characterization of the strains revealed that such prophage enrichment is detectable only in exDNA samples and would likely be missed in whole-genome DNA preparations (e.g., detection of episomal prophages did not correlate with higher prophage excision rates nor higher excised prophage copy numbers in qPCR experiments using whole-genome DNA). In S. aureus MSSA476, we found that enrichment and excision of the prophage ϕSa4ms into the cytoplasm was temporal and that episomal prophage localization did not appear to be a precursor to lytic cycle replication, suggesting ϕSa4ms excision into the cytoplasm may be part of a novel lysogenic switch. For example, we show that ϕSa4ms excision alters the promoter and transcription of htrA2, encoding a stress-response serine protease, and that alternative promotion of htrA2 confers increased heat-stress survival in S. aureus COL. Overall, exDNA isolation and focused sequencing may offer a more complete genomic picture for bacterial pathogens, offering insights into important chromosomal dynamics likely missed with whole-genome DNA-based approaches.

Many genomes are incorrectly identified at GenBank. We developed a plan to find and correct misidentified genomes using genomic comparison statistics together with a scaffold of reliably identified genomes from type. A workshop was organized with broad representation from the bacterial taxonomic community to review the proposal, the GenBank Microbial Genomic Taxonomy Workshop, Bethesda MD, May 12–13, 2015. Electronic supplementary material The online version of this article (doi:10.1186/s40793-016-0134-1) contains supplementary material, which is available to authorized users.