SAMSA: a comprehensive metatranscriptome analysis pipeline

BackgroundAlthough metatranscriptomics-the study of diverse microbial population activity based on RNA-seq data-is rapidly growing in popularity, there are limited options for biologists to analyze this type of data. Current approaches for processing metatranscriptomes rely on restricted databases and a dedicated computing cluster, or metagenome-based approaches that have not been fully evaluated for processing metatranscriptomic datasets. We created a new bioinformatics pipeline, designed specifically for metatranscriptome dataset analysis, which runs in conjunction with Metagenome-RAST (MG-RAST) servers. Designed for use by researchers with relatively little bioinformatics experience, SAMSA offers a breakdown of metatranscriptome transcription activity levels by organism or transcript function, and is fully open source. We used this new tool to evaluate best practices for sequencing stool metatranscriptomes.ResultsWorking with the MG-RAST annotation server, we constructed the Simple Annotation of Metatranscriptomes by Sequence Analysis (SAMSA) software package, a complete pipeline for the analysis of gut microbiome data. SAMSA can summarize and evaluate raw annotation results, identifying abundant species and significant functional differences between metatranscriptomes. Using pilot data and simulated subsets, we determined experimental requirements for fecal gut metatranscriptomes. Sequences need to be either long reads (longer than 100 bp) or joined paired-end reads. Each sample needs 40-50 million raw sequences, which can be expected to yield the 5-10 million annotated reads necessary for accurate abundance measures. We also demonstrated that ribosomal RNA depletion does not equally deplete ribosomes from all species within a sample, and remaining rRNA sequences should be discarded. Using publicly available metatranscriptome data in which rRNA was not depleted, we were able to demonstrate that overall organism transcriptional activity can be measured using mRNA counts. We were also able to detect significant differences between control and experimental groups in both organism transcriptional activity and specific cellular functions.ConclusionsBy making this new pipeline publicly available, we have created a powerful new tool for metatranscriptomics research, offering a new method for greater insight into the activity of diverse microbial communities. We further recommend that stool metatranscriptomes be ribodepleted and sequenced in a 100 bp paired end format with a minimum of 40 million reads per sample

Native solitary bee reproductive success depends on early season precipitation and host plant richness

Spring-emerging bees depend upon the synchronized bloom times of angiosperms that provide pollen and nectar for offspring. The emergence of such bees and bloom times are linked to weather but can be phenologically mismatched, which could limit bee developmental success. However, it remains unclear how such phenologically asynchrony could affect spring-emerging pollinators, and especially for those that forage over a relatively short time period. We examined the relationship between weather and host plant selection on the native spring-foraging solitary bee, Osmia lignaria, across three years at urban and rural sites in and around Seattle, Washington. We used community science weather data to test the effects of precipitation, wind, and temperature on O. lignaria oviposition and developmental success. We also collected pollen data over two distinct foraging periods, early and late spring, and used Next-Generation Sequencing to identify plant genera from pollen. Among the weather variables, precipitation during the early foraging period adversely affected larval developmental success and adult bee emergence success, but not oviposition. Using DNA metabarcoding, we observed that increases in the number of plant genera in pollen increased adult emergence in both foraging periods, but not oviposition or larval development. We also observed that foraging bees consistently visited certain genera during each foraging period, especially Acer, Salix, and Rubus. However, pollen collected by O. lignaria over different years varied in the number of total genera visited, highlighting the importance of multi-year studies to ascertain bee foraging preferences and its link to developmental success.USDA-NIFA McIntire Stennis Cooperative Forestry Program-Accession No. 1012774 (to PCT) and the University of Washington Hall Conservation Genetics Fund (to LRW

SAMSA2: a standalone metatranscriptome analysis pipeline

Abstract Background Complex microbial communities are an area of growing interest in biology. Metatranscriptomics allows researchers to quantify microbial gene expression in an environmental sample via high-throughput sequencing. Metatranscriptomic experiments are computationally intensive because the experiments generate a large volume of sequence data and each sequence must be compared with reference sequences from thousands of organisms. Results SAMSA2 is an upgrade to the original Simple Annotation of Metatranscriptomes by Sequence Analysis (SAMSA) pipeline that has been redesigned for standalone use on a supercomputing cluster. SAMSA2 is faster due to the use of the DIAMOND aligner, and more flexible and reproducible because it uses local databases. SAMSA2 is available with detailed documentation, and example input and output files along with examples of master scripts for full pipeline execution. Conclusions SAMSA2 is a rapid and efficient metatranscriptome pipeline for analyzing large RNA-seq datasets in a supercomputing cluster environment. SAMSA2 provides simplified output that can be examined directly or used for further analyses, and its reference databases may be upgraded, altered or customized to fit the needs of any experiment

Fecal metatranscriptomics of macaques with idiopathic chronic diarrhea reveals altered mucin degradation and fucose utilization

Abstract Background Idiopathic chronic diarrhea (ICD) is a common cause of morbidity and mortality among juvenile rhesus macaques. Characterized by chronic inflammation of the colon and repeated bouts of diarrhea, ICD is largely unresponsive to medical interventions, including corticosteroid, antiparasitic, and antibiotic treatments. Although ICD is accompanied by large disruptions in the composition of the commensal gut microbiome, no single pathogen has been concretely identified as responsible for the onset and continuation of the disease. Results Fecal samples were collected from 12 ICD-diagnosed macaques and 12 age- and sex-matched controls. RNA was extracted for metatranscriptomic analysis of organisms and functional annotations associated with the gut microbiome. Bacterial, fungal, archaeal, protozoan, and macaque (host) transcripts were simultaneously assessed. ICD-afflicted animals were characterized by increased expression of host-derived genes involved in inflammation and increased transcripts from bacterial pathogens such as Campylobacter and Helicobacter and the protozoan Trichomonas. Transcripts associated with known mucin-degrading organisms and mucin-degrading enzymes were elevated in the fecal microbiomes of ICD-afflicted animals. Assessment of colon sections using immunohistochemistry and of the host transcriptome suggests differential fucosylation of mucins between control and ICD-afflicted animals. Interrogation of the metatranscriptome for fucose utilization genes reveals possible mechanisms by which opportunists persist in ICD. Bacteroides sp. potentially cross-fed fucose to Haemophilus whereas Campylobacter expressed a mucosa-associated transcriptome with increased expression of adherence genes. Conclusions The simultaneous profiling of bacterial, fungal, archaeal, protozoan, and macaque transcripts from stool samples reveals that ICD of rhesus macaques is associated with increased gene expression by pathogens, increased mucin degradation, and altered fucose utilization. The data suggest that the ICD-afflicted host produces fucosylated mucins that are leveraged by potentially pathogenic microbes as a carbon source or as adhesion sites

Fecal metatranscriptomics of macaques with idiopathic chronic diarrhea reveals altered mucin degradation and fucose utilization.

BACKGROUND:Idiopathic chronic diarrhea (ICD) is a common cause of morbidity and mortality among juvenile rhesus macaques. Characterized by chronic inflammation of the colon and repeated bouts of diarrhea, ICD is largely unresponsive to medical interventions, including corticosteroid, antiparasitic, and antibiotic treatments. Although ICD is accompanied by large disruptions in the composition of the commensal gut microbiome, no single pathogen has been concretely identified as responsible for the onset and continuation of the disease. RESULTS:Fecal samples were collected from 12 ICD-diagnosed macaques and 12 age- and sex-matched controls. RNA was extracted for metatranscriptomic analysis of organisms and functional annotations associated with the gut microbiome. Bacterial, fungal, archaeal, protozoan, and macaque (host) transcripts were simultaneously assessed. ICD-afflicted animals were characterized by increased expression of host-derived genes involved in inflammation and increased transcripts from bacterial pathogens such as Campylobacter and Helicobacter and the protozoan Trichomonas. Transcripts associated with known mucin-degrading organisms and mucin-degrading enzymes were elevated in the fecal microbiomes of ICD-afflicted animals. Assessment of colon sections using immunohistochemistry and of the host transcriptome suggests differential fucosylation of mucins between control and ICD-afflicted animals. Interrogation of the metatranscriptome for fucose utilization genes reveals possible mechanisms by which opportunists persist in ICD. Bacteroides sp. potentially cross-fed fucose to Haemophilus whereas Campylobacter expressed a mucosa-associated transcriptome with increased expression of adherence genes. CONCLUSIONS:The simultaneous profiling of bacterial, fungal, archaeal, protozoan, and macaque transcripts from stool samples reveals that ICD of rhesus macaques is associated with increased gene expression by pathogens, increased mucin degradation, and altered fucose utilization. The data suggest that the ICD-afflicted host produces fucosylated mucins that are leveraged by potentially pathogenic microbes as a carbon source or as adhesion sites