Next-generation sequencing is becoming instrumental in global transcriptome analysis as it provides deeper coverage and higher throughput, making it possible to better understand complex diseases such as cancer. In turn, the SOLiD® System and other next-generation sequencing platforms are being used to more accurately quantify RNA expression levels using the platform’s respective RNA-Seq library preparation methodology. However, not all RNA-Seq analyses yield the same level of accuracy. Outside of inherent differences in platforms, the library preparation method can influence sequencing results and can be the difference between a novel discovery and an incorrect conclusion.

Strand Orientation: Figure 1Enlarge Image
Figure 1. Schematic showing the possible reads that arise when strand orientation is preserved or is not preserved. Click to enlarge.

Not all RNA-Seq library preparation methods are equivalent. Standard methods for constructing an RNA-Seq library fail to distinguish which strand a transcript is produced from. Consequently, sequencing data show equal levels of transcription from both strands. This highlights the need to preserve strand orientation throughout the library construction process. Ambion®, the leader in RNA analysis technologies, offers the first kit to streamline high-throughput sequencing of the whole transcriptome and small RNAs, while maintaining strand orientation of a particular transcript represented in the resulting short sequence reads. That is, all mapped reads are aligned in the direction of transcription relative to the chromosomal strand. By convention, the chromosomal strand is indicated by ‘+’ or ‘-‘ and is annotated as such in major genome browsers and databases.

In practice, one can confirm that strand orientation is preserved when all reads align in the 5’ to 3’ orientation of an annotated exon or transcript. For example, if the library preparation method preserves strand orientation, and gene 1 is on the ‘+’ strand of a particular chromosome, all sequence reads will only align to the gene 1 exons on the ‘+’ strand. If the library does not preserve the strand orientation, an equally distributed mixture of ‘+’ and ‘-‘ strand reads are aligned to exons for gene 1. This means that both 5’→3’ oriented reads as well as reads that are the reverse complement of gene 1 exons are aligned. Consequently, the ‘+/-‘ read output can can be ambiguous when the researcher attempts to measure transcript levels in situations where there are two proximate (or overlapping) transcripts from both strands of the chromosome, or where there is antisense transcription. These situations mask the true transcriptome picture, and information that is important in the elucidation of disease profiles and regulatory functions is lost.

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