Optimization of de novo transcriptome assembly from high-throughput short read sequencing data improves functional annotation for non-model organisms

Background: The k-mer hash length is a key factor affecting the output of de novo transcriptome assembly packages using de Bruijn graph algorithms. Assemblies constructed with varying single k-mer choices might result in the loss of unique contiguous sequences (contigs) and relevant biological information. A common solution to this problem is the clustering of single k-mer assemblies. Even though annotation is one of the primary goals of a transcriptome assembly, the success of assembly strategies does not consider the impact of k-mer selection on the annotation output. This study provides an in-depth k-mer selection analysis that is focused on the degree of functional annotation achieved for a non-model organism where no reference genome information is available. Individual k-mers and clustered assemblies (CA) were considered using three representative software packages. Pair-wise comparison analyses (between individual k-mers and CAs) were produced to reveal missing Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog identifiers (KOIs), and to determine a strategy that maximizes the recovery of biological information in a de novo transcriptome assembly. Results: Analyses of single k-mer assemblies resulted in the generation of various quantities of contigs and functional annotations within the selection window of k-mers (k-19 to k-63). For each k-mer in this window, generated assemblies contained certain unique contigs and KOIs that were not present in the other k-mer assemblies. Producing a non-redundant CA of k-mers 19 to 63 resulted in a more complete functional annotation than any single k-mer assembly. However, a fraction of unique annotations remained (~0.19 to 0.27% of total KOIs) in the assemblies of individual k-mers (k-19 to k-63) that were not present in the non-redundant CA. A workflow to recover these unique annotations is presented. Conclusions: This study demonstrated that different k-mer choices result in various quantities of unique contigs per single k-mer assembly which affects biological information that is retrievable from the transcriptome. This undesirable effect can be minimized, but not eliminated, with clustering of multi-k assemblies with redundancy removal. The complete extraction of biological information in de novo transcriptomics studies requires both the production of a CA and efforts to identify unique contigs that are present in individual k-mer assemblies but not in the CA

Human Occupancy as a Source of Indoor Airborne Bacteria

Exposure to specific airborne bacteria indoors is linked to infectious and noninfectious adverse health outcomes. However, the sources and origins of bacteria suspended in indoor air are not well understood. This study presents evidence for elevated concentrations of indoor airborne bacteria due to human occupancy, and investigates the sources of these bacteria. Samples were collected in a university classroom while occupied and when vacant. The total particle mass concentration, bacterial genome concentration, and bacterial phylogenetic populations were characterized in indoor, outdoor, and ventilation duct supply air, as well as in the dust of ventilation system filters and in floor dust. Occupancy increased the total aerosol mass and bacterial genome concentration in indoor air PM10 and PM2.5 size fractions, with an increase of nearly two orders of magnitude in airborne bacterial genome concentration in PM10. On a per mass basis, floor dust was enriched in bacterial genomes compared to airborne particles. Quantitative comparisons between bacterial populations in indoor air and potential sources suggest that resuspended floor dust is an important contributor to bacterial aerosol populations during occupancy. Experiments that controlled for resuspension from the floor implies that direct human shedding may also significantly impact the concentration of indoor airborne particles. The high content of bacteria specific to the skin, nostrils, and hair of humans found in indoor air and in floor dust indicates that floors are an important reservoir of human-associated bacteria, and that the direct particle shedding of desquamated skin cells and their subsequent resuspension strongly influenced the airborne bacteria population structure in this human-occupied environment. Inhalation exposure to microbes shed by other current or previous human occupants may occur in communal indoor environments

Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation

Background: The lack of sequenced genomes for oleaginous microalgae limits our understanding of the mechanisms these organisms utilize to become enriched in triglycerides. Here we report the de novo transcriptome assembly and quantitative gene expression analysis of the oleaginous microalga Neochloris oleoabundans, with a focus on the complex interaction of pathways associated with the production of the triacylglycerol (TAG) biofuel precursor. Results: After growth under nitrogen replete and nitrogen limiting conditions, we quantified the cellular content of major biomolecules including total lipids, triacylglycerides, starch, protein, and chlorophyll. Transcribed genes were sequenced, the transcriptome was assembled de novo, and the expression of major functional categories, relevant pathways, and important genes was quantified through the mapping of reads to the transcriptome. Over 87 million, 77 base pair high quality reads were produced on the Illumina HiSeq sequencing platform. Metabolite measurements supported by genes and pathway expression results indicated that under the nitrogen-limiting condition, carbon is partitioned toward triglyceride production, which increased fivefold over the nitrogen-replete control. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during nitrogen limitation was bolstered by repression of the β-oxidation pathway, up-regulation of genes encoding for the pyruvate dehydrogenase complex which funnels acetyl-CoA to lipid biosynthesis, activation of the pentose phosphate pathway to supply reducing equivalents to inorganic nitrogen assimilation and fatty acid biosynthesis, and the up-regulation of lipases—presumably to reconstruct cell membranes in order to supply additional fatty acids for TAG biosynthesis. Conclusions: Our quantitative transcriptome study reveals a broad overview of how nitrogen stress results in excess TAG production in N. oleoabundans, and provides a variety of genetic engineering targets and strategies for focused efforts to improve the production rate and cellular content of biofuel precursors in oleaginous microalgae.Connecticut Center for Advanced Technology (Fuel Diversification Grant, by the National Science Foundation Grant #0854322)Yale Climate and Energy InstituteYale Institute for Biospheric StudiesYale University. Biomedical High Performance Computing CenterNational Institutes of Health (U.S.) (NIH Grant # RR19895

Human occupancy as a source of indoor airborne bacteria.

Exposure to specific airborne bacteria indoors is linked to infectious and noninfectious adverse health outcomes. However, the sources and origins of bacteria suspended in indoor air are not well understood. This study presents evidence for elevated concentrations of indoor airborne bacteria due to human occupancy, and investigates the sources of these bacteria. Samples were collected in a university classroom while occupied and when vacant. The total particle mass concentration, bacterial genome concentration, and bacterial phylogenetic populations were characterized in indoor, outdoor, and ventilation duct supply air, as well as in the dust of ventilation system filters and in floor dust. Occupancy increased the total aerosol mass and bacterial genome concentration in indoor air PM(10) and PM(2.5) size fractions, with an increase of nearly two orders of magnitude in airborne bacterial genome concentration in PM(10). On a per mass basis, floor dust was enriched in bacterial genomes compared to airborne particles. Quantitative comparisons between bacterial populations in indoor air and potential sources suggest that resuspended floor dust is an important contributor to bacterial aerosol populations during occupancy. Experiments that controlled for resuspension from the floor implies that direct human shedding may also significantly impact the concentration of indoor airborne particles. The high content of bacteria specific to the skin, nostrils, and hair of humans found in indoor air and in floor dust indicates that floors are an important reservoir of human-associated bacteria, and that the direct particle shedding of desquamated skin cells and their subsequent resuspension strongly influenced the airborne bacteria population structure in this human-occupied environment. Inhalation exposure to microbes shed by other current or previous human occupants may occur in communal indoor environments

Enrichment of bacteria in airborne particulate matter and floor dust.

<p>Bacterial mass percentage (100×bacterial mass divided by total particle mass) in indoor air, outdoor air, and duct supply air samples and in the PM_2.5 and PM₁₀ size fraction of resuspended floor dust samples. Mass fractions were estimated assuming an average mass of 655 fg per bacterium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034867#pone.0034867-Ilic1" target="_blank">[25]</a>. Box and whisker plots have the same interpretation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034867#pone-0034867-g001" target="_blank">Figure 1</a>.</p

The influence of floor dust resuspension and particle shedding on particle number concentrations of varying optical diameter.

<p>Plotted are the ratio of occupied indoor to simultaneous outdoor particle number concentrations for five size ranges from 0.3 µm to 10 µm under the following three conditions. Black bars represent the case of 30 people sitting on a carpeted floor that is covered with plastic sheeting (to prevent resuspension of floor dust). White bars represent one person walking on a carpeted floor covered with plastic sheeting. Gray bars represent one person walking on a carpeted floor (without plastic sheeting). Error bars indicate one standard error of the mean for replicate experiments. The experiment in which 30 people were sitting on a carpeted floor covered with plastic sheeting was conducted only once.</p

Airborne particulate matter, filter dust, and floor dust samples acquired and analyzed in this study.

<p>Airborne particulate matter, filter dust, and floor dust samples acquired and analyzed in this study.</p

Relative abundances of bacteria in the indoor air, ventilation duct air, floor dust, and HVAC filter dust samples.

<p>Relative abundances of the 20 most common bacterial taxa in indoor air, ventilation duct air, HVAC filter dust, and floor dust. Indoor and ventilation duct air include PM₁₀ samples from indoor air when the room was occupied. Floor dust samples were sieved PM₃₇ floor dust and resuspended PM₁₀ floor dust taken after occupancy. HVAC filter dust represents samples from the filter of the building HVAC system that handled a variable mixture of outdoor air and indoor return air. Taxa are classified to the highest taxonomic level to which they could be confidently assigned. Error bars represent one standard error of the mean for nine indoor air PM₁₀ samples, four floor dust samples, and three HVAC duct samples. Groups shown represent 55% of floor dust, 83% of HVAC filter dust, 51% of indoor air taxa, and 46% of ventilation duct air taxa.</p