Novel bioinformatic pipeline for de novo transcriptomics analysis: application in a Patagonian native tree species (Nothofagus pumilio)

ESTRAVIS BARCALÁ, M.; ARANA, M.V.; MARCHELLI, P; HEER, K.; ZIEGENHAGEN, B; BELLORA, N.

Rosario

Simposio; Simposio de Genómica Funcional de Plantas, Rosario, Argentina; 2017

CCT CONICET Rosario

Nothofagus pumilio (common name: Lenga) is the most abundant tree species of the template Patagonian forest, the southernmost woody ecosystem on Earth. Despite its ecological and economical importance, physiological and molecular studies in N. pumilio are scarce, and the genomic resources for the whole genus are very limited (only a partial draft transcriptome for N. nervosa, a related species). Given our interest in the overall effect of high temperatures in N. pumilio?s physiology in a context of climate change, we performed NGS on leaf poly-A-selected RNA from plants exposed to contrasting temperatures (20° and 34°C) in a growth chamber controlled environment. Two biological replicates from independent experiments were sequenced for each temperature. The non-model nature of this species implied a de novo assembly and all downstream analysis with no reference genome. In this talk we present some problems of great interest to the ?non-model plant species community? and how we managed to overcome them with a hands-on programming approach. We will show an original bioinformatic pipeline that integrates de novo transcriptome assembly and transcriptome mapping, with both standard and new tools developed in our lab aimed to solve basic problems faced by working without a reference genome, such as miss-assembled contigs, sequence redundancy and strand specificity. Besides, this pipeline allows the annotation against a custom database (in our case, 12 broad-leaf tree transcriptomes from an open-access website, http://www.hardwoodgenomics.org/). Finally, we will show differential expression and transcriptome-based phylogeny results using our corrected transcriptome.