Researchers at Harvard University have developed a method of truly digital RNA-Seq that does not rely on the counting of individual reads. How is this possible you ask?
Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, paired-end deep sequencing is performed to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. The barcodes have been optimized to be unambiguously identifiable, even in the presence of multiple sequencing errors.
With this method, they have developed a simple strategy for mitigating sequence-dependent bias and inaccuracy at low copy numbers, inherent to standard RNA-Seq library preparation techniques.
- Shiroguchi K, Jia TZ, Sims PA, Xie XS ( 2012) Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. PNAS [Epub ahead of print]. [abstract]