Whole transcriptome analysis with sequencing: methods, challenges and potential solutions

Abstract

Whole transcriptome analysis plays an essential role in deciphering genome structure and function, identifying genetic networks underlying cellular, physiological, biochemical and biological systems and establishing molecular biomarkers that respond to diseases, pathogens and environmental challenges. Here, we review transcriptome analysis methods and technologies that have been used to conduct whole transcriptome shotgun sequencing or whole transcriptome tag/target sequencing analyses. We focus on how adaptors/linkers are added to both 5' and 3' ends of mRNA molecules for cloning or PCR amplification before sequencing. Challenges and potential solutions are also discussed. In brief, next generation sequencing platforms have accelerated releases of the large amounts of gene expression data. It is now time for the genome research community to assemble whole transcriptomes of all species and collect signature targets for each gene/transcript, and thus use known genes/transcripts to determine known transcriptomes directly in the near future.

Fig. 1

Whole transcriptome shotgun sequencing. a Lists both EST and RNA-seq procedures, while b, c demonstrate library preparations for full-length cDNA and 3′-directed cDNA sequencing. EST, full-length cDNA and 3′-directed cDNA are all cloning-based approaches using Sanger sequencing so that the adaptors are provided by the cloning vectors. However, the adaptors used in RNA-seq library preparation depend on the next generation sequencing platforms. Certainly other derivatives exist for whole transcriptome shotgun sequencing or RNA-seq

Fig. 2

Whole transcriptome target/tag sequencing with restriction digestion. SAGE, MPSS, DGETP and TALEST share many common steps in library preparation: both anchoring (AE) and tagging enzymes (TE) are used with adaptors added in slightly different ways. In PAT-A, rSAGE and 3′ end cDNA amplification, only the anchor enzyme cut site is used for 5′ adaptor ligation, while the 3′ adaptor/linker is combined with reverse transcription. For 3′ most target/tag collection, only 3′ end cDNA amplification uses specific primers, while the remaining methods rely on magnetic bead selection

Fig. 3

SAGE-seq with multiple enzymes. The process represents a combination of SAGE and DDRT-PCR methods as the 5′ prolonged region was adapted from the former, while the 3′ prolonged region from the latter technique, respectively. Adaptors for the 5′ prolonged regions are designed according to the enzyme cut sites: Tsp509I (AATT), NlaIII (CATG), MspI (CCGG) and DpnII (GATC)

Fig. 4

Whole transcriptome target/tag sequencing without restriction digestion (I). 3PC, PATs and 3′READS represent methods with enrichment of fragmented polyA+ RNA to conduct polyA site sequencing. These methods share common steps including size selection, PCR amplification and next generation sequencing

Fig. 5

Whole transcriptome target/tag sequencing without restriction digestion (II). 3′T-fill and EXPRSS represent methods with enrichment of fragmented polyA+ cDNA to conduct polyA site sequencing. These methods share common steps including size selection, PCR amplification and next generation sequencing

Fig. 6

Whole transcriptome target/tag sequencing without restriction digestion (III). PAS-seq and PolyA-seq represent methods with custom primers containing dTs at 3′ end to conduct polyA site sequencing. These methods share common steps including size selection, PCR amplification and next generation sequencing