Abstract

Most modern methods for NGS library prep require the use of enzyme processing, such as DNA polymerase reactions, which can introduce errors in the form of incorrect sequence and misrepresented copy number. Conventional RNA sequencing library construction involves the ligation of a population of cDNA molecules with adapters prior to amplification and sequencing. An inherent weakness of conventional RNA-Seq analysis is that cDNA fragments that amplify more efficiently will unavoidably result in a higher number of reads than cDNAs that do not amplify as well during the library construction PCR step. Therefore, when multiple reads mapping to the same transcript are encountered, it is not possible to determine whether sequenced reads originate from the same or different cDNA molecules. With Molecular Indexed™ libraries, each molecule is tagged with a molecular index randomly chosen from ~10,000 combinations so that any two identical molecules become distinguishable (with odds of 10,000/1), and can be independently evaluated in later data analysis. Analysis using molecular indexing information provides an absolute, digital measurement of gene expression levels, irrespective of common amplification distortions observed in many RNA-Seq experiments. This type of indexing requires no additional steps in RNA-Seq workflow and increases the precision of downstream analysis. 

 At low sequencing depths, analysis using qRNA-Seq is identical to conventional analysis and generates equivalent RPKM values in all applications. As sequencing depth increases, individual molecular resolution also increases. In quantitative RNA-Seq experiments, the molecular indices distinguish re-sampling of the same molecule from sampling of a different molecule. At high sequencing depths, each molecule can be distinguished and the entire library can be analyzed to provide absolute numbers of each molecule. Resolving individual clones of molecules is critical for increasing sequen