Angela M Yu | bioRxiv Science

bioRxiv2015

Characterizing RNA structures in vitro and in vivo with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

RNA molecules adopt a wide variety of structures that perform many cellular functions, including catalysis, small molecule sensing, and cellular defense, among others. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

Biochemistry↗

bioRxiv2015

Nanopore Sequencing in Microgravity

The ability to perform remote, in situ sequencing and diagnostics has been a long-sought goal for point-of-care medicine and portable DNA/RNA measurements. This technological advancement extends to missions beyond Earth as well, both for crew health and astrobiology applications. However, most commercially available sequencing technologies are ill-suited for space flight for a variety of reasons, including excessive volume and mass, and insufficient ruggedization for spaceflight. Portable and lightweight nanopore-based sequencers, which analyze nucleic acids electrochemically, are inherently much better suited to spaceflight, and could potentially be incorporated into future missions with only minimal modification. As a first step toward evaluating the performance of nanopore sequencers in a microgravity environment, we tested the Oxford Nanopore Technologies MinION in a parabolic flight simulator to examine the effect of reduced gravity on DNA sequencing. The instrument successfully generated three reads, averaging 2,371 bases. However, the median current was shifted across all reads and the error profiles changed compared with operation of the sequencer on the ground, indicating that distinct computational methods may be needed for such data. We evaluated existing methods and propose two new methods; the first new method is based on a wave-fingerprint method similar to that of the Shazam model for matching periodicity information in music, and the second is based on entropy signal mapping. These tools provide a unique opportunity for nucleic acid sequencing in reduced gravity environments. Finally, we discuss the lessons learned from the parabolic flight as they would apply to performing DNA sequencing with the MinION aboard the International Space Station.

Genomics↗