Evaluation of the impact of RNA preservation methods of spiders for de novo transcriptome assembly

Abstract

With advances in high-throughput sequencing technologies, de novo transcriptome sequencing and assembly has become a cost-effective method to obtain comprehensive genetic information of a species of interest, especially in nonmodel species with large genomes such as spiders. However, high-quality RNA is essential for successful sequencing, and sample preservation conditions require careful consideration for the effective storage of field-collected samples. To this end, we report a streamlined feasibility study of various storage conditions and their effects on de novo transcriptome assembly results. The storage parameters considered include temperatures ranging from room temperature to -80°C; preservatives, including ethanol, RNAlater, TRIzol and RNAlater-ICE; and sample submersion states. As a result, intact RNA was extracted and assembly was successful when samples were preserved at low temperatures regardless of the type of preservative used. The assemblies as well as the gene expression profiles were shown to be robust to RNA degradation, when 30 million 150-bp paired-end reads are obtained. The parameters for sample storage, RNA extraction, library preparation, sequencing and in silico assembly considered in this work provide a guideline for the study of field-collected samples of spiders.

A novel method for single whitefly (Bemisia tabaci) transcriptomes reveals an eleven amino acid deletion in theNusGprotein in the bacterial endosymbiontPortiera aleyrodidaru

“…Across all transcriptomes over 30M reads were obtained. The amount of transcripts were comparable to those reported in other arthropoda studies from field collections [34]. However, we did not observe any difference in assembly qualities as did [34]; probably due to the fact that our field-collected samples had degraded RNA based on RIN, and thus direct comparison with [34] was inappropriate.…”

“…However, to date all transcriptome sequencing has involved pooled samples obtained through rearing several generations of isolines of a single species to ensure high quantities of RNA for subsequent sequencing. This remains a major bottle neck in particular within arthropoda where collected samples are limited due to small morphological sizes [33, 34]. In addition, the development of isolines is time consuming and often has colonies dying off mainly due to inbreeding depression [35].…”

Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

“…Finally, long-term storage temperature is confounded with liquid nitrogen and RNAlater treatments in our study and long-term storage temperature is known to drive RNA integrity (Gayral et al, 2013;Kono et al, 2016). Our goal was to replicate typical field experiments, where reliable refrigeration is not available for substantial amounts of time, and RNAlater is used as the predominant preservation method.…”

“…Fry at 30 days postfertilization (dpf) were <5 mm long, transparent and highly permeable. To mimic field conditions, RNAlater individuals were stored at room temperature for 17 days (Camacho-Sanchez et al, 2013;Kono, Nakamura, Ito, Tomita, & Arakawa, 2016). Procedures for all experiments performed were approved by the Institutional Animal Care and Use Committee at Florida Atlantic University (Protocol #A15-32).…”

RNAlater and flash freezing storage methods nonrandomly influence observed gene expression in RNAseq experiments

“…RNAlater treatments in our study and long-term storage temperature is known to drive 379 RNA integrity (Kono et al 2016) (Gayral et al 2013 Figure S3: Box plots depicting GC content of genes that were differentially expressed 573 between treatments (e.g. "Higher exp.…”