Comparison of next generation sequencing technologies for transcriptome characterization

Abstract

BackgroundWe have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis.ResultsThe Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics.ConclusionNG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms.

Harnessing Next Generation Sequencing in Climate Change: RNA-Seq Analysis of Heat Stress-Responsive Genes in Wheat (Triticum aestivum L.)

“…under the HS. Very high levels of upregulation of RCA-A has been reported in response to HS in wheat (Wan et al, 2008), which is concurrent with our observations. Different mechanisms involving signaling molecules, chaperones, TFs, and other SAGs has been reported to be the reason behind the wide-thermotolerance diversity in wheat (Qin et al, 2008), similar to our present findings.…”

“…HD2329 under the control and HS-treated conditions. Earlier, Roche GS-FLX 454 was the most widely used platform for de novo transcriptome sequencing, due to its long read length in different organisms, for example, ginseng (Sun et al, 2010), A. thaliana (Wall et al, 2009), and maize (Vega-Arreguin et al, 2009). The Illumina transcriptome was mainly used for the sequencing of organisms whose genome was sequenced .…”

“…With a view to maximize annotation percentage, five different databases (NCBI nonredundant, UniProt: Swiss-Prot, TriFLDB, PlantCyc, and Pfam) were used, as reported earlier by Wu et al (2014) for de novo transcriptome of common bean. Previous studies have identified approximately 165,499 unigenes in wheat infested with Fusarium blight , and 14,550 DETs in wheat at the anthesis period (Wan et al, 2008). DETs in response to inorganic phosphate (P i ) deprivation were identified using de novo assembly in wheat (Misson et al, 2005).…”

Applications of next generation sequencing in molecular ecology of non-model organisms

“…Nevertheless, normalized cDNA libraries have still been useful in some cases to this end (for example, Schwarz et al, 2009;Ekblom et al, 2010). When planning the experiment it is also valuable to note that there is a trade off between the cost of normalization and the cost of sequencing (Wall et al, 2009). As sequencing technology keeps improving and the quantity of sequence yield increases, cDNA normalization may soon be superfluous for transcriptome characterization, as a single sequencing run will be able to cover the complete transcriptome of the sampled tissue, regardless of the distribution of transcript abundances.…”

“…However, 454 sequencing presents slightly higher rates of sequencing errors compared with competing technologies (see Table 1), especially in homopolymers (see Glossary) (Huse et al, 2007). Simulation experiments have shown that when there is no reference genome available, a combination of different NGS technologies may be most cost effective for transcriptome characterization (Wall et al, 2009). …”