Going Long and Direct

Abstract

Biologist Chris Vollmers of the University of California, Santa Cruz relies on RNA sequencing to study the rich and unique biology of B cells, where each cell lineage recombines its genome to produce specific antibodies. As these cells modulate their response to immune challenges, they can further mutate and produce diverse transcripts, even within the same individuals. To study these cells, short-read RNA sequencing, the current gold standard, fell short.After reverse transcription, Vollmers' team produced fulllength cDNA that they then chopped into short sequences of roughly 200 bases in length. "You have this beautiful full-length isoform information that you then lose completely because of the technology in the library preparation," Vollmers said. The resulting data was cumbersome to assemble, limiting his ability to study transcriptional isoforms and the coding regions for antibodies.So in a recent study (1), his team tried a long-read strategy. They took advantage of the full-length cDNA and used Oxford Nanopore's MinION sequencer to examine whole sequences in a single read. They soon realized that there was far more going on in B cells than they could see with short-read data. "You think you have a homogenous population, but really, cells make totally different isoforms, which may do totally different things," he said.Vollmer's work is just one example of researchers fueling sequencing innovations for mining the rich biology of RNA. Scientists are also using sequencing to find and study chemical modifications to transcripts. Routine direct RNA sequencing is