June 1, 2021

Library prep and bioinformatics improvements for full-length transcript sequencing on the PacBio Sequel System

Author(s): Vierra, Michelle and Galvin, Brendan and Hon, Ting and Ferroa, Heather and Topfer, Armin and Li, Yuan and Tseng, Elizabeth

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. Here we describe an improved, simplified library workflow and analysis pipeline that reduces library preparation time, RNA input, and cost. The Iso-Seq V2 Express workflow is a one day protocol that requires only ~300 ng of total RNA input while also reducing the number of reverse transcription and amplification steps down to single reactions. Compared with the previous workflow, the Iso-Seq V2 Express workflow increases the percentage of full-length (FL) reads while achieving a higher average transcript length. At the same time, the Iso-Seq 3 analysis recently released in the SMRT Link 6.0 software is a major improvement over previous versions. Iso-Seq 3 is highly accurate at detecting and removing library artifacts (TSO and RT artifacts) as well as differentiating barcodes on multiplexed samples. Iso-Seq 3 achieves the same output performance in high-quality transcript sequences compared to previous versions while reducing the runtime and memory usage dramatically.

Organization: PacBio
Year: 2019

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