Nanopore Whole Transcriptome Analysis and Pathogen Surveillance by a Novel Solid‐Phase Catalysis Approach

Advanced Science

Boce Zhang¹  Manyun Yang²  Daniel Sun³  Aihua Zhang³  Yi Fang³  Amogh Changavi³  Ming‐Qun Xu³  Luo Sun³  Zhiyi Sun³ 

[1] Department of Food Science and Human Nutrition University of Florida Gainesville FL 32603 USA;Department of Microbiology and Immunology Cornell University Ithaca NY 14853 USA;New England Biolabs, Inc. Ipswich MA 01938 USA;

关键词: direct RNA‐seq;    foodborne pathogen;    immobilized enzymes;    next‐generation sequencing;    Oxford Nanopore Technologies;    transcriptome;   

DOI  :  10.1002/advs.202103373

来源: DOAJ

【 摘 要 】

Abstract The requirement of a large input amount (500 ng) for Nanopore direct RNA‐seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid‐phase reversible immobilization (SPRI) beads. A novel solid‐phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here. This new approach leverages concurrent processing of non‐polyadenylated transcripts with immobilized poly(A) polymerase and T4 DNA ligase, followed by directly loading the prepared library onto a flow cell. Whole transcriptome sequencing, using a human pathogen Listeria monocytogenes as a model, demonstrates this new method displays little sample loss, takes much less time, and generates higher sequencing throughput correlated with reduced nanopore fouling compared to the current library preparation for 500 ng input. Consequently, this approach enables Nanopore low‐input direct RNA‐seq, improving pathogen detection and transcript identification in a microbial community standard with spike‐in transcript controls. Besides, as evident in the bioinformatic analysis, the new method provides accurate RNA consensus with high fidelity and identifies higher numbers of expressed genes for both high and low input RNA amounts.

【 授权许可】

Unknown