What Factors Jeopardize Whole Transcriptome Analysis using Next Generation Sequencing Approaches?

Whole transcriptome analysis begins with preparation of next generation sequencing libraries from high quality total RNA or purified mRNA samples, followed by sequencing using the state-of-art high throughput sequencers and data processing and analysis.  During development of our whole transcriptome target sequencing (WTTS) method to capture 3’ ends of transcripts, we observed several factors that jeopardized whole transcriptome analysis.  Inappropriate primer design resulted in both recessive and dominant “amplification detours” that produced noisy reads and biased data.  In addition, excessive PCR cycles and high concentrations of primers resulted in over-amplification of next generation sequencing libraries and reduced transcriptome coverage.  Incomplete genome sequencing and partial gene annotation results in missing data, particularly when a 3’ end sequencing approach is employed.  Overlapping genes can transfer reads from one expressed gene to another non-expressed gene or cause non-strand-restricted reads (such as paired-end reads) to be unmapped. RNA-seq tends to enlarge whole transcriptome up-bottom boundaries, producing a much longer list of differentially expressed genes in comparison to the WTTS method. Furthermore, RNA-seq reads cannot be used to detect polyA sites or potential isoform switches in tissues at different time points. When an Illumina analyzer is used for sequencing, libraries with low diversity must be avoided.  We will demonstrate cases associated with these challenges at the Workshop.