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. 2009 Oct;37(18):e123.
doi: 10.1093/nar/gkp596. Epub 2009 Jul 20.

Transcriptome analysis by strand-specific sequencing of complementary DNA

Dmitri Parkhomchuk  1 , Tatiana Borodina, Vyacheslav Amstislavskiy, Maria Banaru, Linda Hallen, Sylvia Krobitsch, Hans Lehrach, Alexey Soldatov
Affiliations

Transcriptome analysis by strand-specific sequencing of complementary DNA

Dmitri Parkhomchuk et al. Nucleic Acids Res. 2009 Oct.

Abstract

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.

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Figures

Figure 1.
Figure 1.
ssRNA-Seq method. (A) Flowchart of the ssRNA-Seq procedure. RNA is shown in red, DNA in green. Arrows are in the 5′ to 3′ direction. (B–E) Scatter plots comparing mouse mRNA expression data (number of reads in annotated genes). (B) The same mouse liver sample, strand-specific (ssRNA-Seq, X-axis) and strand-unspecific (RNA-Seq, Y-axis) protocols (Pearson correlation coefficient (cc) = 0.999). (C) ssRNA-Seq results for two biological replicas (mouse whole brain mRNA); cc = 0.990. (D) Our mouse whole brain expression data (X-axis) and data from (8) (Y-axis); cc = 0.817. (E) Sense (X-axis) and antisense (Y-axis) expression in mouse brain. (F and G) Overlap of the yeast YGR203W gene with a non-annotated gene in a head-to-head orientation. Transcriptional profile without orientation is shown in (F), with orientation in (G). Reads mapped in the forward direction are shown in blue; in the reverse direction in red. Vertical lines mark the boundaries of the YGR203W gene, as determined previously (6).
Figure 2.
Figure 2.
Cumulative profiles of transcription (blue: sense, pink: antisense) and end tags for sense orientation (red) in 5′ and 3′ regions of mouse and yeast genes. X-axis: positions relative to the 5′ (left panels) or 3′ (right panels) end of the gene; Y-axis: total number of sequencing reads or end tags mapped in this position.
Figure 3.
Figure 3.
Different types of intergenic regions in yeast. Stacked columns show the distribution of 7103 annotated intergenic regions according to orientation and relative position of neighboring genes. The neighboring genes were counted as overlapping if it was impossible to find a 30 nt ‘gap’ (the interval not covered by sequencing reads) between them. Transcription initiation requires more space than transcription termination: genes tend to be closer to each other in a tail-to-tail than in a head-to-head orientation. The mean distances between ORF's are 375 bp in tail-to-tail, 590 bp in head-to-tail and 703 bp in head-to-head orientations. About 49% of 3′-ends (tails) overlap with neighboring genes. This is about two times more than the fraction of overlapping 5′-ends (heads), which is 24%.
Figure 4.
Figure 4.
Cumulative profiles of transcription and sense end tags in 3′ regions of internal exons for mouse (left) and yeast (right).
See this image and copyright information in PMC

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