Comparative Transcriptomic Approaches Exploring Contamination Stress Tolerance in Salix sp. Reveal the Importance for a Metaorganismal de Novo Assembly Approach for Nonmodel Plants

Abstract

Metatranscriptomic study of nonmodel organisms requires strategies that retain the highly resolved genetic information generated from model organisms while allowing for identification of the unexpected. A real-world biological application of phytoremediation, the field growth of 10 Salix cultivars on polluted soils, was used as an exemplar nonmodel and multifaceted crop response well-disposed to the study of gene expression. Sequence reads were assembled de novo to create 10 independent transcriptomes, a global transcriptome, and were mapped against the Salix purpurea 94006 reference genome. Annotation of assembled contigs was performed without a priori assumption of the originating organism. Global transcriptome construction from 3.03 billion paired-end reads revealed 606,880 unique contigs annotated from 1588 species, often common in all 10 cultivars. Comparisons between transcriptomic and metatranscriptomic methodologies provide clear evidence that nonnative RNA can mistakenly map to reference genomes, especially to conserved regions of common housekeeping genes, such as actin, a/b-tubulin, and elongation factor 1-a. In Salix, Rubisco activase transcripts were down-regulated in contaminated trees across all 10 cultivars, whereas thiamine thizole synthase and CP12, a Calvin Cycle master regulator, were uniformly up-regulated. De novo assembly approaches, with unconstrained annotation, can improve data quality; care should be taken when exploring such plant genetics to reduce de facto data exclusion by mapping to a single reference genome alone. Salix gene expression patterns strongly suggest cultivar-wide alteration of specific photosynthetic apparatus and protection of the antenna complexes from oxidation damage in contaminated trees, providing an insight into common stress tolerance strategies in a real-world phytoremediation system.Coppiced willows have the ability to produce high biomass yields in temperate regions under challenging conditions and have positive impacts on biodiversity (Labrecque et al

ANCHOR: a 16S rRNA gene amplicon pipeline for microbial analysis of multiple environmental samples

“…ANCHOR is designed with utility for nonideal, uncharacterised biology in mind and, in particular, to provide flexibility to complex data sets and complement high uncertainty metatranscriptomics (Gonzalez et al ., ; Brereton et al ., ; Gonzalez et al ., ). While benchmarking against synthetic or simple (mock) communities is essential, an equally important test of the technology is its utility to contribute to unknown biology in complex real‐world systems.…”

“…Default recommended annotation uses four sequence repositories with strict BLASTn criteria (>99% identity and coverage) providing each amplicon with up to four pools of labels from: NCBI‐curated bacterial and Archaea RefSeq, NCBI nr/nt, SILVA and Ribosomal Database Project (RDP). Annotation selection against the four databases is based on de novo metatranscriptomics strategy (Brereton et al ., ; Gonzalez et al ., ), where all potential annotation is retained to allow for informed annotation selection and downstream interpretation. BLASTn returns with a query identity and coverage <99% are discarded and the highest identity/coverage scores are selected per query.…”

“…BLASTn returns with a query identity and coverage <99% are discarded and the highest identity/coverage scores are selected per query. When the highest identity/coverage for a given high‐count sequence is shared amongst different blast returns from a database, all are retained as equally ‘good’ annotation and designated as ambiguous hits (borrowed from the idea of secondary annotation in metatranscriptomics [Brereton et al ., ; Gonzalez et al ., )]. Accurately reporting ambiguity is important as fragments of a specific 16S rRNA gene [or a gene's entire sequence (Vetrovsky and Baldrian, )] are sometimes 100% similar between known species; the relative utility of a specific amplicon as a barcode therefore varies based on the species present in a sample as well as the technology used for sequencing and data processing.…”

Trees, fungi and bacteria: tripartite metatranscriptomics of a root microbiome responding to soil contamination

“…E values < 10 −4 (protein blast) and < 10 −6 (nucleotide blast) were used as cutoffs. A previously reported method for selecting annotation (based on the percentage of maximum potential bitscore) from blastx returns was used to help select the primary annotation given multiple high scoring alignments for a single sequence, all statistically characterised as non-random [ 2 ]. BLAST hits that were not selected but have a high comparable percentage of maximum potential bitscore (within 10%) were retained for each contig as (alternative) secondary annotation.…”

“…The observation of gene expression across multiple interacting organisms has the potential to better reflect the complex reality of biology than the observation of organisms in isolation [ 1 ]. By separating the assembly of RNA sequence data from annotation (identification) of assembled contigs, de novo metatranscriptome assembly allows for such observation without a prerequisite for, and therefore bias from, reference genome sequences from organisms expected to be present within any biological system [ 2 , 3 ]. A metatranscriptomic approach designed without constraint to any a priori defined organism, but open to annotation from any sequenced strata of life, should be powerful in biological systems already recognised as highly complex, such as the human digestive tract or rhizosphere microbiome (although such microbiome complexity could arguably be defined by the current extent of study in a biological field).…”