PT  - JOURNAL ARTICLE
AU  - Longhua Guo
AU  - James Boocock
AU  - Jacob M. Tome
AU  - Sukantha Chandrasekaran
AU  - Evann E. Hilt
AU  - Yi Zhang
AU  - Laila Sathe
AU  - Xinmin Li
AU  - Chongyuan Luo
AU  - Sriram Kosuri
AU  - Jay A. Shendure
AU  - Valerie A. Arboleda
AU  - Jonathan Flint
AU  - Eleazar Eskin
AU  - Omai B. Garner
AU  - Shangxin Yang
AU  - Joshua S. Bloom
AU  - Leonid Kruglyak
AU  - Yi Yin
TI  - Rapid cost-effective viral genome sequencing by V-seq
AID  - 10.1101/2020.08.15.252510
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.08.15.252510
4099  - http://biorxiv.org/content/early/2020/08/15/2020.08.15.252510.short
4100  - http://biorxiv.org/content/early/2020/08/15/2020.08.15.252510.full
AB  - Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, require extensive sample handling time, and have limited throughput. Here, we describe V-seq, an inexpensive, fast, and scalable method that performs targeted viral genome sequencing by multiplexing virus-specific primers at the cDNA synthesis step. We designed densely tiled reverse transcription (RT) primers across the SARS-CoV-2 genome, with a subset of hexamers at the 3’ end to minimize mis-priming from the abundant human rRNA repeats and human RNA PolII transcriptome. We found that overlapping RT primers do not interfere, but rather act in concert to improve viral genome coverage in samples with low viral load. We provide a path to optimize V-seq with SARS-CoV-2 as an example. We anticipate that V-seq can be applied to investigate genome evolution and track outbreaks of RNA viruses in a cost-effective manner. More broadly, the multiplexed RT approach by V-seq can be generalized to other applications of targeted RNA sequencing.Competing Interest StatementSK is employed by and hold equity, JSB consults for and holds equity in Octant Inc.