Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows

Stefan Siebert,Freya Goetz,Mark D Robinson,Steven H D Haddock,Sophia C Tintori,Stephen A Smith,Casey W Dunn,Rebecca R Helm,Nathan Shaner,Johannes Jaeger

doi:10.1371/journal.pone.0022953

Abstract

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing.

Highlights

Siphonophores belong to Cnidaria, a diverse group of animals that includes corals, Hydra, and jellyfish
The Newbler assembly consists of 9,471 genes which include a combined total of 13,727 contigs. 1,007 of the isogroups had multiple contigs and multiple splice variants consisting of different combinations of contigs
There are a variety of off-the-shelf workflows for analyzing gene expression with next-generation sequencing tools. These workflows are multi-step processes that differ in sample preparation, sequencing methods, and mapping tools

Summary

Introduction

Siphonophores belong to Cnidaria, a diverse group of animals that includes corals, Hydra, and jellyfish. There have been no studies of differential gene expression between functionally specialized zooids in siphonophores. Such analyses would help identify genes that specify zooid types, and play a role in the development and functions of different zooid phenotypes. In sequencing-based expression studies, fragments of transcripts are sequenced and the resulting reads are mapped to known gene reference sequences. The number of reads that map to each gene sequence in the reference provides a measure of its expression level [10,11]. NGS expression studies have been largely limited to model species because their well-annotated genomes provide high quality references for mapping [11,12]. There is, growing interest in using these tools to quantify expression in non-model species

Methods

Results

Conclusion