Passow, C. N. | bioRxiv Science

bioRxiv2018

Performance of gene expression analyses using de novo assembled transcripts in polyploid species

MotivationQuality of gene expression analyses using de novo assembled transcripts in species experienced recent polyploidization is yet unexplored.\n\nResultsFive plant species with various polyploidy history were used for differential gene expression (DGE) analyses. DGE analyses using putative genes inferred by Trinity performed similar to or better than Corset and Grouper in precision, but lower in sensitivity. In species that lack polyploidy event in the past few million years, DGE analyses using de novo assembled transcriptome identified 50-76% of the differentially expressed genes recovered by mapping reads to the reference genes. However, in species with more recent polyploidy event, the percentage decreased to 7-30%. In addition, 7-89% of differentially expressed genes from de novo assembly are contaminations. Gene co-expression network analyses using de novo assemblies vs. mapping to the reference genes recovered the same module that significantly correlated with treatment in one of the five species tested.\n\nAvailability and ImplementationCommands and scripts used in this study are available at https://bitbucket.org/lychen83/chen_et_al_2018_benchmark_dge/; Analysis files are available at Dryad doi: XXXXXX.\n\nContactlychen83@qq.com\n\nSupplementary informationSupplementary data are available at Bioinformatics online

bioinformatics↗

bioRxiv2018

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

RNA-sequencing is a popular next-generation sequencing technique for assaying genome-wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field-based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer-term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome-wide expression patterns in 30 day old fry collected from the same brood at the same time point that were flash-frozen in liquid nitrogen and stored at -80{degrees}C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash-freezing relative to RNAlater-storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field-based studies are substantial and should not be ignored.

genomics↗