Microbial communities are commonly characterized by amplifying and sequencing target genes, but errors limit the precision of amplicon sequencing. We present DADA2, a software package that models and corrects amplicon errors. DADA2 identified more real variants than other methods in Illumina-sequenced mock communities, some differing by a single nucleotide, while outputting fewer spurious sequences. DADA2 analysis of vaginal samples revealed a diversity of Lactobacillus crispatus strains undetected by OTU methods.The importance of microbial communities to human and environmental health has motivated methods for their efficient characterization. The most common, and cost-effective, method is the amplification and sequencing of targeted genetic elements. Amplicon sequencing of taxonomic marker genes such as 16S rRNA [1], the ITS region [2] or 18S rRNA [3] provides a census of a community. Functional diversity can be probed by targeting functional genes [4].Disentangling errors from biological variation in amplicon sequencing data presents unique challenges, which has prompted the development of amplicon-specific error-correction methods [5,6,7,8]. Most of these methods were designed for pyrosequenced amplicons, and cannot be applied to Illumina sequencing.Currently, errors in Illumina-sequenced amplicon data are most often addressed by filtering low quality reads and constructing Operational Taxonomic Units (OTUs): clusters of sequences that differ by less than a fixed dissimilarity threshold (typically 3%) within which sequence variation is ignored [9,10,11]. Lumping similar sequences together reduces the rate at which errors are misinterpreted as biological variation, but OTUs under-utilize the quality of modern sequencing by precluding the possibility of resolving fine-scale (or strain-level ) variation [7,12,13,14,15]. Recent studies have shown that fine-scale variation can be informative about ecological niches [12,13], temporal dynamics [15], and population structure [4]. Fine-scale variation differentiates pathogenic from commensal strains in some cases [16,17], and can contain clinically relevant information for more complex microbiome-associated diseases [18,19,20].DADA -the Divisive Amplicon Denoising Algorithm -was introduced to correct pyrosequenced amplicon errors without constructing OTUs [7]. DADA was shown to identify real variation at the finest scales in 454-sequenced amplicon data while outputting few false positives [7,4].Here we present DADA2, an extension and reimplementation of DADA adapted for use with Illumina sequencing and available as an open-source R package available at https: //github.com/benjjneb/dada2. DADA2 implements a new model of Illumina-sequenced amplicon errors that incorporates quality information. Banded alignments and a kmerdistance screen improve computational performance. The DADA2 R package provides light-weight tools for other key parts of the amplicon denoising workflow: filtering, derepli-1 cation, chimera identification, and merging paired-end reads.We compared DAD...

SummaryCost, time, and expertise constraints limit traditional observation-based comprehensive biodiversity assessment. Therefore, surrogate focal taxa representative of wider biodiversity are commonly used as an imperfect ‘proxy’. Contemporary biodiversity assessments are also increasingly benefiting from the combination of high-throughput sequencing and metagenomic methodologies that enable identification of environmental DNA samples. However, there is a need for empirical studies combining the use of surrogate taxa with metagenomic approaches, that promise rapid and efficient biodiversity assessment.We here tested for the first time the possibility of using the intestinal contents of wild-collected dung beetles (Scarabaeidae) as a source of mammalian DNA, in a metagenomics proof-of-concept approach to directly detect and identify mammals from an area of savanna-scrub in southern Africa. Dung beetles have been purveyed as an indirect proxy measure of mammalian diversity, owing to their dependence upon vertebrate dung as a food source, and the ease with which they can be comprehensively sampled using simple and repeatable trapping protocols, achievable much faster than vertebrate surveys.Following shotgun sequencing of gut content DNA extractions from ten dung beetle species, we used in silico filters to identify mammals by searching the resulting reads against known mammalian mitochondrial DNA from online sequence repositories, matching 546 paired reads to known mitogenomes held in GenBank, and 634 reads to known mammal barcode sequences held in BOLD. Identified mammalian sequences were consistent with wild and domesticated ungulates known from the sampling site, and included blue wildebeest, plains zebra, and domestic cattle and goat. Four dung beetle samples yielded sufficient sequence data to successfully assemble the near-complete mitogenome of blue wildebeest at up to 21 X mean coverage, despite low initial DNA concentrations, unambiguously corroborating identification.It is conceptually and practically possible to rapidly and economically apply metagenomic techniques in dung beetle gut sequencing to detect the presence of mammals upon whose dung the beetles have fed. Since the approach can be readily scaled up, it may prove to be of practical use as a complement to traditional biodiversity assessment methods, and should be tested in usefulness for detecting rare, endangered or cryptic mammal species.

Eight Polled Dorset lambs were orally inoculated with Sarcocystis ovicanis sporocysts. Two lambs that received 100,000 or 200,000 sporocysts became clinically ill, recovered, and were killed 67 and 88 days after inoculation (DAI). Numerous intramuscular cysts were found in their skeletal and cardiac muscles. Three lambs received 100,000 sporocysts, three lambs received 1 million sporocysts, and three lambs received no sporocysts. After an acute clinical illness characterized by anemia, inappetence, weight loss, fever, and reduced serum protein, all lambs that received 100,000 sporocysts died 27 to 29 DAI and all that received 1 million sporocysts died 24 or 25 DAI. Hemorrhage involving the striated muscle and visceral organs was the most apparent gross lesion. The heart appeared most severely affected. Schizonts were found in vascular endothelial cells of all six inoculated lambs. Uninoculated lambs remained healthy, and neither lesions nor parasites were found in any tissues. Dogs fed tissues containing S. ovicanis cysts produced sporocytes 11 to 37 days after feeding; cats fed similar stages produced no sporocysts. Dogs fed tissues containing schizonts produced no sporocysts.

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Individual sporocysts containing 4 sporozoites were shed in the feces of dogs, foxes, and raccoons after ingestion of bovine tissue infected with Sarcocystis fusiformis bradyzoites. No sporocysts were shed by cats, monkeys, swine, skunks, ferrets, rats, guinea pigs, or rabbits after ingestion of similar bovine tissue. The shedding of sporocysts by dogs that had ingested tissue from a bovine experimentally infected with sporocysts from coyotes indicated that both canids were definitive hosts for the same species of Sarcocystis. After oral inoculation wtih sporocysts from dogs previously fed infected bovine heart, a calf became infected; but sheep, swine, rats, mice, rabbits, and monkeys did not. These results show the narrow specificity of the asexual stages of this parasite for the bovine intermediate host and the wider, though still restricted, host range of the sexual stages of this parasite for coyotes, dogs, foxes, and raccoons, the definitive hosts.

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A generally nonlethal Salmonella typhimurium infection in weanling rats produced bacterial myocarditis and myocardial hyperplasia. Myocardial lesions were characterized by focal infiltrates of inflammatory cells (predominantly mononuclear). segmental myocyte necrosis, and incipient fibrosis. Although bacterial infections are infrequently associated with myocarditis, the S. typhimurium infection in young rats produced a new experimental model of diffuse myocardial inflammatory foci. Biochemical changes in the myocardium included great increases in total myocardial contents of protein (23%), RNA (39%) and DNA (43%) and several lipid fractions (35–55%) as well as in tissue activities of acid hydrolases, such as cathepsin D (124%) and β‐glucuronidase (135%), all of which contrasted with the relatively limited areas of histologic involvement (1.5%). To study the effects of additional stress in this model infection, some rats were exercised by forced running in wheels for 2 hours and others were fasted for 24 hours before samples were obtained. The short period of forced exercise in this infection caused an additional increase of myocardial protein content (47%) but with no additional change in histology. The expected fasting‐induced degradation of protein as well as an infection‐associated increase in myocardial lipids were each prevented when rats were fasted during ongoing acute infection. Protein degradation, as reflected by heightened acid hydrolase activities, seemed to occur at a similar rate regardless of other stresses, whereas the rate of myocardial protein synthesis appeared to be alterable.

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