Next generation sequencing (NGS) technologies are a rapidly developing area, with the ability to produce gigabases of sequencing data at reduced costs. While NGS is best recognized for genomic DNA sequencing (DNA-Seq), there is a significant body of work to expand the breadth of sample types being studied. RNA deep sequencing (RNA-Seq) is a well-developed segment of NGS experimentation, with a growing focus on small RNA deep sequencing (smRNA-Seq). One of the limitations in the deep sequencing of the 20-30 nt small RNA is in the sample preparation workflow, which requires several purification steps. As with all RNA samples, NGS sample preparation involves the ligation of fixed sequences, called adapters, onto the 5? and 3? ends of the starting RNA library. This ligation step can be plagued by the undesired joining of the adapter sequences to one another without a segment of the library in between, resulting in adapter dimer formation. Although affinity capture can be used to remove adapter dimers in most library prep workflows, adapter-tagged small RNA libraries are too close in size to the adapter dimers for effective use of this approach. As result, a gel purification step is required, which can deplete low abundance sequences. To avoid making unintentional changes to the complexity of the library, it is advantageous to block adapter dimer formation at the ligation step. With this goal in mind, we propose a novel approach to RNA sample preparation for NGS that uses TriLink's expertise in nucleic acid chemistry to disfavor adapter-adapter ligation while allowing for efficient joining of adapter probes on the 5'and 3'ends of the library.
Next generation sequencing (NGS) is maturing into an essential technique for RNA and DNA sequence analysis. NGS is a major component of the genomics market which was valued at $5.8 billion in 2009. We propose the investigation of chemically-modified components to streamline NGS experimental workflows for small RNA deep sequencing.
|Khnouf, Ruba; Shore, Sabrina; Han, Crystal M et al. (2018) Efficient Production of On-Target Reads for Small RNA Sequencing of Single Cells Using Modified Adapters. Anal Chem 90:12609-12615|