ASAP 2: a pipeline and web server to analyze marker gene amplicon sequencing data automatically and consistently.

BACKGROUND: Amplicon sequencing of marker genes such as 16S rDNA have been widely used to survey and characterize microbial community. However, the complex data analyses have required many interfering manual steps often leading to inconsistencies in results. RESULTS: Here, we have developed a pipeline, amplicon sequence analysis pipeline 2 (ASAP 2), to automate and glide through the processes without the usual manual inspections and user's interference, for instance, in the detection of barcode orientation, selection of high-quality region of reads, and determination of resampling depth and many more. The pipeline integrates all the analytical processes such as importing data, demultiplexing, summarizing read profiles, trimming quality, denoising, removing chimeric sequences and making the feature table among others. The pipeline accepts multiple file formats as input including multiplexed or demultiplexed, paired-end or single-end, barcode inside or outside and raw or intermediate data (e.g. feature table). The outputs include taxonomic classification, alpha/beta diversity, community composition, ordination analysis and statistical tests. ASAP 2 supports merging multiple sequencing runs which helps integrate and compare data from different sources (public databases and collaborators). CONCLUSIONS: Our pipeline minimizes hands-on interference and runs amplicon sequence variant (ASV)-based amplicon sequencing analysis automatically and consistently. Our web server assists researchers that have no access to high performance computer (HPC) or have limited bioinformatics skills. The pipeline and web server can be accessed at https://github.com/tianrenmaogithub/asap2 and https://hts.iit.edu/asap2 , respectively.

Major transitions in dinoflagellate evolution unveiled by phylotranscriptomics.

Dinoflagellates are key species in marine environments, but they remain poorly understood in part because of their large, complex genomes, unique molecular biology, and unresolved in-group relationships. We created a taxonomically representative dataset of dinoflagellate transcriptomes and used this to infer a strongly supported phylogeny to map major morphological and molecular transitions in dinoflagellate evolution. Our results show an early-branching position of Noctiluca, monophyly of thecate (plate-bearing) dinoflagellates, and paraphyly of athecate ones. This represents unambiguous phylogenetic evidence for a single origin of the group's cellulosic theca, which we show coincided with a radiation of cellulases implicated in cell division. By integrating dinoflagellate molecular, fossil, and biogeochemical evidence, we propose a revised model for the evolution of thecal tabulations and suggest that the late acquisition of dinosterol in the group is inconsistent with dinoflagellates being the source of this biomarker in pre-Mesozoic strata. Three distantly related, fundamentally nonphotosynthetic dinoflagellates, Noctiluca, Oxyrrhis, and Dinophysis, contain cryptic plastidial metabolisms and lack alternative cytosolic pathways, suggesting that all free-living dinoflagellates are metabolically dependent on plastids. This finding led us to propose general mechanisms of dependency on plastid organelles in eukaryotes that have lost photosynthesis; it also suggests that the evolutionary origin of bioluminescence in nonphotosynthetic dinoflagellates may be linked to plastidic tetrapyrrole biosynthesis. Finally, we use our phylogenetic framework to show that dinoflagellate nuclei have recruited DNA-binding proteins in three distinct evolutionary waves, which included two independent acquisitions of bacterial histone-like proteins.

Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates.

Plastid establishment involves the transfer of endosymbiotic genes to the host nucleus, a process known as endosymbiotic gene transfer (EGT). Large amounts of EGT have been shown in several photosynthetic lineages but also in present-day plastid-lacking organisms, supporting the notion that endosymbiotic genes leave a substantial genetic footprint in the host nucleus. Yet the extent of this genetic relocation remains debated, largely because the long period that has passed since most plastids originated has erased many of the clues to how this process unfolded. Among the dinoflagellates, however, the ancestral peridinin-containing plastid has been replaced by tertiary plastids on several more recent occasions, giving us a less ancient window to examine plastid origins. In this study, we evaluated the endosymbiotic contribution to the host genome in two dinoflagellate lineages with tertiary plastids. We generated the first nuclear transcriptome data sets for the "dinotoms," which harbor diatom-derived plastids, and analyzed these data in combination with the available transcriptomes for kareniaceans, which harbor haptophyte-derived plastids. We found low level of detectable EGT in both dinoflagellate lineages, with only 9 genes and 90 genes of possible tertiary endosymbiotic origin in dinotoms and kareniaceans, respectively, suggesting that tertiary endosymbioses did not heavily impact the host dinoflagellate genomes.

Targeted Next-Generation Sequencing Assay for Direct Detection and Serotyping of Salmonella from Enrichment.

In this study, an automated, targeted next-generation sequencing (tNGS) assay to detect and serotype Salmonella from sample enrichments was evaluated. The assay generates millions of reads to detect multiple Salmonella-specific genes and serotype-specific alleles, detecting all Salmonella spp. tested to date, and serotyping 62 common Salmonella serotypes. Accuracy was tested on 291 pure reference cultures (251 Salmonella, 40 non-Salmonella), 21 artificially contaminated poultry carcass rinse samples, and 363 naturally contaminated poultry environmental samples. Among the 291 pure reference cultures, the automated tNGS assay resulted in 100% detection accuracy, 100% serotyping accuracy for the claimed serotypes, and 0% false positives. The limit of detection was estimated at 5 × 104 CFU/mL by testing enumerated cultures of strains representative of six serotypes. In cocontamination studies with mixtures of two serotypes (Enteritidis, Typhimurium, Kentucky, Infantis, and Newport) at a 1:1 ratio, tNGS detected both serotypes with 100% accuracy. The assay demonstrated 100% accuracy in artificially contaminated poultry carcass rinse sample enrichments. Targeted NGS was highly effective in detecting Salmonella in samples collected from poultry production facilities. Results demonstrated that tNGS could detect Salmonella and provide accurate serotyping information consistent with conventional serology. These findings highlight the reliable and efficient performance of a fully automated tNGS Salmonella assay in detecting and identifying Salmonella strains in complex matrices, reducing the time to results from 4 to 5 days required by the traditional isolation and serotyping to 10-12 h for tNGS after primary enrichment.

A regulatory toolbox of MiniPromoters to drive selective expression in the brain.

The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.

VBCG: 20 validated bacterial core genes for phylogenomic analysis with high fidelity and resolution.

BACKGROUND: Phylogenomic analysis has become an inseparable part of studies of bacterial diversity and evolution, and many different bacterial core genes have been collated and used for phylogenomic tree reconstruction. However, these genes have been selected based on their presence and single-copy ratio in all bacterial genomes, leaving out the gene's 'phylogenetic fidelity' unexamined. RESULTS: From 30,522 complete genomes covering 11,262 species, we examined 148 bacterial core genes that have been previously used for phylogenomic analysis. In addition to the gene presence and single-copy rations, we evaluated the gene's phylogenetic fidelity by comparing each gene's phylogeny with its corresponding 16S rRNA gene tree. Out of the 148 bacterial genes, 20 validated bacterial core genes (VBCG) were selected as the core gene set with the highest bacterial phylogenetic fidelity. Compared to the larger gene set, the 20-gene core set resulted in more species having all genes present and fewer species with missing data, thereby enhancing the accuracy of phylogenomic analysis. Using Escherichia coli strains as examples of prominent bacterial foodborne pathogens, we demonstrated that the 20 VBCG produced phylogenies with higher fidelity and resolution at species and strain levels while 16S rRNA gene tree alone could not. CONCLUSION: The 20 validated core gene set improves the fidelity and speed of phylogenomic analysis. Among other uses, this tool improves our ability to explore the evolution, typing and tracking of bacterial strains, such as human pathogens. We have developed a Python pipeline and a desktop graphic app (available on GitHub) for users to perform phylogenomic analysis with high fidelity and resolution. Video Abstract.

The Light Chain Domain and Especially the C-Terminus of Receptor-Binding Domain of the Botulinum Neurotoxin (BoNT) Are the Hotspots for Amino Acid Variability and Toxin Type Diversity.

Botulinum neurotoxins (BoNT) are the most potent toxins in the world. They are produced by a few dozens of strains within several clostridial species. The toxin that they produce can cause botulism, a flaccid paralysis in humans and other animals. With seven established serologically different types and over 40 subtypes, BoNTs are among the most diverse known toxins. The toxin, its structure, its function and its physiological effects on the neural cell and animal hosts along with its diversity have been the subjects of numerous studies. However, many gaps remain in our knowledge about the BoNT toxin and the species that produce them. One of these gaps involves the distribution and extent of variability along the full length of the gene and the protein as well as its domains and subdomains. In this study, we performed an extensive analysis of all of the available 143 unique BoNT-encoding genes and their products, and we investigated their diversity and evolution. Our results indicate that while the nucleotide variability is almost uniformly distributed along the entire length of the gene, the amino acid variability is not. We found that most of the differences were concentrated along the protein's light chain (LC) domain and especially, the C-terminus of the receptor-binding domain (HCC). These two regions of the protein are thus identified as the main source of the toxin type differentiation, and consequently, this toxin's versatility to bind different receptors and their isoforms and act upon different substrates, thus infecting different hosts.

The power, potential, benefits, and challenges of implementing high-throughput sequencing in food safety systems.

The development and application of modern sequencing technologies have led to many new improvements in food safety and public health. With unprecedented resolution and big data, high-throughput sequencing (HTS) has enabled food safety specialists to sequence marker genes, whole genomes, and transcriptomes of microorganisms almost in real-time. These data reveal not only the identity of a pathogen or an organism of interest in the food supply but its virulence potential and functional characteristics. HTS of amplicons, allow better characterization of the microbial communities associated with food and the environment. New and powerful bioinformatics tools, algorithms, and machine learning allow for development of new models to predict and tackle important events such as foodborne disease outbreaks. Despite its potential, the integration of HTS into current food safety systems is far from complete. Government agencies have embraced this new technology, and use it for disease diagnostics, food safety inspections, and outbreak investigations. However, adoption and application of HTS by the food industry have been comparatively slow, sporadic, and fragmented. Incorporation of HTS by food manufacturers in their food safety programs could reinforce the design and verification of effectiveness of control measures by providing greater insight into the characteristics, origin, relatedness, and evolution of microorganisms in our foods and environment. Here, we discuss this new technology, its power, and potential. A brief history of implementation by public health agencies is presented, as are the benefits and challenges for the food industry, and its future in the context of food safety.

Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense.

At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

The dinoflagellates Durinskia baltica and Kryptoperidinium foliaceum retain functionally overlapping mitochondria from two evolutionarily distinct lineages.

BACKGROUND: The dinoflagellates Durinskia baltica and Kryptoperidinium foliaceum are distinguished by the presence of a tertiary plastid derived from a diatom endosymbiont. The diatom is fully integrated with the host cell cycle and is so altered in structure as to be difficult to recognize it as a diatom, and yet it retains a number of features normally lost in tertiary and secondary endosymbionts, most notably mitochondria. The dinoflagellate host is also reported to retain mitochondrion-like structures, making these cells unique in retaining two evolutionarily distinct mitochondria. This redundancy raises the question of whether the organelles share any functions in common or have distributed functions between them. RESULTS: We show that both host and endosymbiont mitochondrial genomes encode genes for electron transport proteins. We have characterized cytochrome c oxidase 1 (cox1), cytochrome oxidase 2 (cox2), cytochrome oxidase 3 (cox3), cytochrome b (cob), and large subunit of ribosomal RNA (LSUrRNA) of endosymbiont mitochondrial ancestry, and cox1 and cob of host mitochondrial ancestry. We show that all genes are transcribed and that those ascribed to the host mitochondrial genome are extensively edited at the RNA level, as expected for a dinoflagellate mitochondrion-encoded gene. We also found evidence for extensive recombination in the host mitochondrial genes and that recombination products are also transcribed, as expected for a dinoflagellate. CONCLUSION: Durinskia baltica and K. foliaceum retain two mitochondria from evolutionarily distinct lineages, and the functions of these organelles are at least partially overlapping, since both express genes for proteins in electron transport.

Horizontal gene transfer and redundancy of tryptophan biosynthetic enzymes in dinotoms.

A tertiary endosymbiosis between a dinoflagellate host and diatom endosymbiont gave rise to "dinotoms," cells with a unique nuclear and mitochondrial redundancy derived from two evolutionarily distinct eukaryotic lineages. To examine how this unique redundancy might have affected the evolution of metabolic systems, we investigated the transcription of genes involved in biosynthesis of the amino acid tryptophan in three species, Durinskia baltica, Kryptoperidinium foliaceum, and Glenodinium foliaceum. From transcriptome sequence data, we recovered two distinct sets of protein-coding transcripts covering the entire tryptophan biosynthetic pathway. Phylogenetic analyses suggest a diatom origin for one set of the proteins, which we infer to be expressed in the endosymbiont, and that the other arose from multiple horizontal gene transfer events to the dinoflagellate ancestor of the host lineage. This is the first indication that these cells retain redundant sets of transcripts and likely metabolic pathways for the biosynthesis of small molecules and extend their redundancy to their two distinct nuclear genomes.

Evidence for the retention of two evolutionary distinct plastids in dinoflagellates with diatom endosymbionts.

Dinoflagellates harboring diatom endosymbionts (termed "dinotoms") have undergone a process often referred to as "tertiary endosymbiosis"--the uptake of algae containing secondary plastids and integration of those plastids into the new host. In contrast to other tertiary plastids, and most secondary plastids, the endosymbiont of dinotoms is distinctly less reduced, retaining a number of cellular features, such as their nucleus and mitochondria and others, in addition to their plastid. This has resulted in redundancy between host and endosymbiont, at least between some mitochondrial and cytosolic metabolism, where this has been investigated. The question of plastidial redundancy is particularly interesting as the fate of the host dinoflagellate plastid is unclear. The host cytosol possesses an eyespot that has been postulated to be a remnant of the ancestral peridinin plastid, but this has not been tested, nor has its possible retention of plastid functions. To investigate this possibility, we searched for plastid-associated pathways and functions in transcriptomic data sets from three dinotom species. We show that the dinoflagellate host has indeed retained genes for plastid-associated pathways and that these genes encode targeting peptides similar to those of other dinoflagellate plastid-targeted proteins. Moreover, we also identified one gene encoding an essential component of the dinoflagellate plastid protein import machinery, altogether suggesting the presence of a functioning plastid import system in the host, and by extension a relict plastid. The presence of the same plastid-associated pathways in the endosymbiont also extends the known functional redundancy in dinotoms, further confirming the unusual state of plastid integration in this group of dinoflagellates.

Tertiary endosymbiosis in two dinotoms has generated little change in the mitochondrial genomes of their dinoflagellate hosts and diatom endosymbionts.

BACKGROUND: Mitochondria or mitochondrion-derived organelles are found in all eukaryotes with the exception of secondary or tertiary plastid endosymbionts. In these highly reduced systems, the mitochondrion has been lost in all cases except the diatom endosymbionts found in a small group of dinoflagellates, called 'dinotoms', the only cells with two evolutionarily distinct mitochondria. To investigate the persistence of this redundancy and its consequences on the content and structure of the endosymbiont and host mitochondrial genomes, we report the sequences of these genomes from two dinotoms. METHODOLOGY/PRINCIPAL FINDINGS: The endosymbiont mitochondrial genomes of Durinskia baltica and Kryptoperidinium foliaceum exhibit nearly identical gene content with other diatoms, and highly conserved gene order (nearly identical to that of the raphid pennate diatom Fragilariopsis cylindrus). These two genomes are differentiated from other diatoms' by the fission of nad11 and by an insertion within nad2, in-frame and unspliced from the mRNA. Durinskia baltica is further distinguished from K. foliaceum by two gene fusions and its lack of introns. The host mitochondrial genome in D. baltica encodes cox1 and cob plus several fragments of LSU rRNA gene in a hugely expanded genome that includes numerous pseudogenes, and a trans-spliced cox3 gene, like in other dinoflagellates. Over 100 distinct contigs were identified through 454 sequencing, but intact full-length genes for cox1, cob and the 5' exon of cox3 were present as a single contig each, suggesting most of the genome is pseudogenes. The host mitochondrial genome of K. foliaceum was difficult to identify, but fragments of all the three protein-coding genes, corresponding transcripts, and transcripts of several LSU rRNA fragments were all recovered. CONCLUSIONS/SIGNIFICANCE: Overall, the endosymbiont and host mitochondrial genomes in the two dinotoms have changed surprisingly little from those of free-living diatoms and dinoflagellates, irrespective of their long coexistence side by side in dinotoms.

The complete plastid genomes of the two 'dinotoms' Durinskia baltica and Kryptoperidinium foliaceum.

BACKGROUND: In one small group of dinoflagellates, photosynthesis is carried out by a tertiary endosymbiont derived from a diatom, giving rise to a complex cell that we collectively refer to as a 'dinotom'. The endosymbiont is separated from its host by a single membrane and retains plastids, mitochondria, a large nucleus, and many other eukaryotic organelles and structures, a level of complexity suggesting an early stage of integration. Although the evolution of these endosymbionts has attracted considerable interest, the plastid genome has not been examined in detail, and indeed no tertiary plastid genome has yet been sequenced. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the complete plastid genomes of two closely related dinotoms, Durinskia baltica and Kryptoperidinium foliaceum. The D. baltica (116470 bp) and K. foliaceum (140426 bp) plastid genomes map as circular molecules featuring two large inverted repeats that separate distinct single copy regions. The organization and gene content of the D. baltica plastid closely resemble those of the pennate diatom Phaeodactylum tricornutum. The K. foliaceum plastid genome is much larger, has undergone more reorganization, and encodes a putative tyrosine recombinase (tyrC) also found in the plastid genome of the heterokont Heterosigma akashiwo, and two putative serine recombinases (serC1 and serC2) homologous to recombinases encoded by plasmids pCf1 and pCf2 in another pennate diatom, Cylindrotheca fusiformis. The K. foliaceum plastid genome also contains an additional copy of serC1, two degenerate copies of another plasmid-encoded ORF, and two non-coding regions whose sequences closely resemble portions of the pCf1 and pCf2 plasmids. CONCLUSIONS/SIGNIFICANCE: These results suggest that while the plastid genomes of two dinotoms share very similar gene content and genome organization with that of the free-living pennate diatom P. tricornutum, the K. folicaeum plastid genome has absorbed two exogenous plasmids. Whether this took place before or after the tertiary endosymbiosis is not clear.

Mitochondrial genome of a tertiary endosymbiont retains genes for electron transport proteins.

Mitochondria and plastids originated through endosymbiosis, and subsequently became reduced and integrated with the host in similar ways. Plastids spread between lineages through further secondary or even tertiary endosymbioses, but mitochondria appear to have originated once and have not spread between lineages. Mitochondria are also generally lost in secondary and tertiary endosymbionts, with the single exception of the diatom tertiary endosymbiont of dinoflagellates like Kryptoperidinium foliaceum, where both host and endosymbiont are reported to contain mitochondria. Here we describe the first mitochondrial genes from this system: cytochrome c oxidase 1 (cox1), cytochrome oxidase 3 (cox3), and cytochrome b (cob). Phylogenetic analyses demonstrated that all characterized genes were derived from the pennate diatom endosymbiont, and not the host. We also demonstrated that all three genes are expressed, that cox1 contains spliced group II introns, and that cob and cox3 form an operon, all like their diatom relatives. The endosymbiont mitochondria not only retain a genome, but also express their genes, and are therefore likely involved in electron transport. Ultrastructural examination confirmed the endosymbiont mitochondria retain normal tubular cristae. Overall, these data suggest the endosymbiont mitochondria have not reduced at the genomic or functional level.