Project description:Purpose: Deconstructing the soil microbiome into reduced-complexity functional modules represents a novel method of microbiome analysis. The goals of this study are to confirm differences in transcriptomic patterns among five functional module consortia. Methods: mRNA profiles of 3 replicates each of functional module enrichments of soil inoculum in M9 media with either 1) xylose, 2) n-acetylglucosamine, 3) glucose and gentamycin, 4) xylan, or 5) pectin were generated by sequencing using an Illumina platform (GENEWIZ performed sequencing). Sequence reads that passed quality filters were aligned to a soil metagenome using Burrows Wheeler Aligner. Resulting SAM files were converted to raw reads using HTSeq, and annotated using Uniref90 or EGGNOG databases. Results: To reduce the size of the RNA-Seq counts table and increase its computational tractability, transcripts containing a minimum of 75 total counts, but no more than 3 zero counts, across the 15 samples were removed. The subsequent dataset was normalized using DESeq2, resulting in a dataset consisting of 6947 unique transcripts across the 15 samples, and 185,920,068 reads. We identified gene categories that were enriched in a sample type relative to the overall dataset using Fisher’s exact test. Conclusions: our dataset confirms that the functional module consortia generated from targeted enrichments of a starting soil inoculum had distinct functional trends by enrichment type.
Project description:Examined soil microbiome microcosms to determine the effect of changing pH on the production of lignocellulolytic enzymes. Soil from a Prosser, Washington field site was incubated in mesh bags on top of a soil interfacing glass bead matrix with MOPS minimal media amended with or without carboxymethyl cellulose at three different pH levels. After incubation, 2 mL of media solution was collected for proteomics, separating intracellular proteome from extracellular proteome via centrifugation. Data was searched with MS-GF+ and MASIC using PNNL's DMS Processing pipeline. Quantitative values were determined using MS1 abundance profiles created by MASIC for every MS2 spectrum, allowing for area under the curve assessments of peptide elution.
Project description:Here we have undertaken a pilot study analysing the proteome of MD2 pineapple using Liquid Chromatography coupled with Mass Spectrometry (LC-MS/MS) and identified 3,818 peptides from 1,781 candidate proteins in the annotated V3 genome sequenced. In addition, a further 603 peptide identifications were found that mapped exclusively to an independent MD2 transcriptome-derived database and were not found in the standard V3 annotated proteome. Peptide identifications derived from the MD2 transcripts were also cross-referenced to a more recent and complete MD2 genome annotation, resulting in 402 non overlapping peptides with either of the genomes. Subsequently, cross-referencing these peptide identifications via their parent transcripts, 30 high-quality gene candidates novel to the V3 pineapple genomes were identified. These analyses add to the knowledge of experimentally validated pineapple genes and demonstrate the utility of transcript-derived proteomics to discover both novel genes and genetic structure in plant genome, and can help improve its annotation.