Challenges and advances for transcriptome assembly in non-model species

Abstract

Analyses of high-throughput transcriptome sequences of non-model organisms are based on two main approaches: de novo assembly and genome-guided assembly using mapping to assign reads prior to assembly. Given the limits of mapping reads to a reference when it is highly divergent, as is frequently the case for non-model species, we evaluate whether using blastn would outperform mapping methods for read assignment in such situations (>15% divergence). We demonstrate its high performance by using simulated reads of lengths corresponding to those generated by the most common sequencing platforms, and over a realistic range of genetic divergence (0% to 30% divergence). Here we focus on gene identification and not on resolving the whole set of transcripts (i.e. the complete transcriptome). For simulated datasets, the transcriptome-guided assembly based on blastn recovers 94.8% of genes irrespective of read length at 0% divergence; however, assignment rate of reads is negatively correlated with both increasing divergence level and reducing read lengths. Nevertheless, we still observe 92.6% of recovered genes at 30% divergence irrespective of read length. This analysis also produces a categorization of genes relative to their assignment, and suggests guidelines for data processing prior to analyses of comparative transcriptomics and gene expression to minimize potential inferential bias associated with incorrect transcript assignment. We also compare the performances of de novo assembly alone vs in combination with a transcriptome-guided assembly based on blastn via simulation and empirically, using data from a cyprinid fish species and from an oak species. For any simulated scenario, the transcriptome-guided assembly using blastn outperforms the de novo approach alone, including when the divergence level is beyond the reach of mapping methods. Combining de novo assembly and a related reference transcriptome for read assignment also addresses the bias/error in contigs caused by the dependence on a related reference alone. Empirical data corroborate those findings when assembling transcriptomes from the two non-model organisms: Parachondrostoma toxostoma (fish) and Quercus pubescens (plant). For the fish species, out of the 31,944 genes known from D. rerio, the guided and de novo assemblies recover respectively 20,605 and 20,032 genes but the performance of the guided assembly approach is much higher for both the contiguity and completeness metrics. For the oak, out of the 29,971 genes known from Vitis vinifera, the transcriptome-guided and de novo assemblies display similar performance but the new guided approach detects 16,326 genes where the de novo assembly only detects 9,385 genes.

Iso-Seq allows genome-independent transcriptome profiling of grape berry development

“…Full-length cDNA sequencing with SMRT technology (Iso-Seq) can be used to rapidly reconstruct the grape berry transcriptome, enabling the identification of cultivar-specific isoforms, refinement of the Cabernet Sauvignon genome annotation, and the creation of a reference for transcriptome-wide expression profiling. In contrast to transcriptome reconstruction using short-read sequencing that requires de novo assembly, Iso-Seq delivers fulllength transcripts that eliminate the introduction of assembly errors and artefacts like chimeric transcripts and incomplete fragments due to PolyA capture (Chang et al 2014;Huang et al 2016;Moreton et al 2016;Smith-Unna et al 2016;Geniza and Jaiswal 2017;Ungaro et al 2017). The incorporation of high-coverage short-read sequencing is still necessary to benefit from the complete transcript sequencing enable by Iso-Seq.…”

Peer Review #2 of "The Oyster River Protocol: a multi-assembler and kmer approach for de novo transcriptome assembly (v0.1)"

“…In addition to reporting synthetic metrics related to assembly structure, TransRate reports individual metrics related to specific elements of assembly quality. One such metric estimates the rate of chimerism, a phenomenon which is known to be problematic in de novo assembly (Ungaro et al, 2017;Singhal, 2013). Rates of chimerism are relatively constant between all assemblers, ranging from 10% for the Shannon assembly, to 12% for the SPAdes75 assembly.…”

“…The ease with which these tools may be used to produce and characterize transcriptome assemblies belies the true complexity underlying the overall process (Ungaro et al, 2017;Wang and Gribskov, 2017;Moreton et al, 2015;Yang and Smith, 2013). Indeed, the subtle (and not so subtle) methodological challenges associated with transcriptome reconstruction may result in highly variable assembly quality.…”

A de novo approach to disentangle partner identity and function in holobiont systems

“…We conclude that the read assignation performed before the assembly 229 step largely contributes to limit the production of chimeras. This shows that the use of SRC_c 230 impacts the de novo assembled transcriptome quality and contributes to address one of the most 231 delicate de novo assembly challenge [43]. The MEGAN6 contigs assignation from M2 shows more 232 contigs than SRC_c could assign to host and symbiont (Tab.…”