Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes

Abstract

Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.

Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns

“…All but one of the 434 transcriptomes used were generated by the One Thousand Plants Project (1KP; www.onekp.com); these transcriptomes were derived from a diverse selection of brown algae, red algae, green algae, bryophytes, lycophytes, ferns, and seed plants (SI Appendix, Table S1). Details on RNA extraction, sequencing, and assembly for 1KP can be found in Johnson et al (40). Additionally, a whole-plant normalized Illumina transcriptome library was constructed and sequenced for Pteridium aquilinum using pooled RNA from six sporophyte tissues (young sporeling leaf, rhizome tip, fiddlehead, mature sterile pinnae, and pinnae with developing and mature sporangia).…”

“…This finding not only provides the first evidence that a plant-to-plant HGT can have a profound evolutionary impact but also has implications for the evolution of photosensory systems in plants. 40 whole or draft genomes of plants and algae (SI Appendix, Table S1) and surprisingly discovered neochrome homologs from hornworts ( Fig. 1B and SI Appendix, Figs.…”

The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves

“…To build the phylogenetic tree, ESV1 and LESV sequences were retrieved from the NCBI and 1000 plants (1KP; Johnson et al, 2012; http://www. onekp.com) databases using BLASTp.…”

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera

“…Furthermore, some names of genes and gene products were updated (see AJ271079.4, EU262887.2, EU262889.2, EU262890.2, and EU262891.2). For Illumina transcriptome sequencing, total RNA extractions, sequencing, and assemblies, see Johnson et al (2012) and Hollister et al (2015).…”