TY - JOUR
T1 - A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses
AU - Reteng, Patrick
AU - Nguyen Thuy, Linh
AU - Tran Thi Minh, Tam
AU - Mares-Guia, Maria Angélica Monteiro de Mello
AU - Torres, Maria Celeste
AU - de Filippis, Ana Maria Bispo
AU - Orba, Yasuko
AU - Kobayashi, Shintaro
AU - Hayashida, Kyoko
AU - Sawa, Hirofumi
AU - Hall, William W.
AU - Nguyen Thi, Lan Anh
AU - Yamagishi, Junya
N1 - Funding Information:
This work was supported by The Japan Society for the Promotion of Science Core-to-Core Program, B. Asia-Africa Science Platforms (id. JPJSCCB20190006), The Atlantic Philanthropies Director Designated Gift Fund (grant id. 27813 and 28091), Faperj under the grant no. E-26/202.930/2016, European Union’s Horizon 2020 grant agreement ZIKACTION no. 734857, Coordenacao de Vigilancia em Saude e Laboratorios de Referencia da Fundacao Oswaldo Cruz/MoH, and World-leading Innovative & Smart Education (WISE) Program Grant-in-Aid for Graduate Student. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.
AB - Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.
UR - http://www.scopus.com/inward/record.url?scp=85115694180&partnerID=8YFLogxK
U2 - 10.1038/s41598-021-98013-9
DO - 10.1038/s41598-021-98013-9
M3 - Article
C2 - 34561471
AN - SCOPUS:85115694180
SN - 2045-2322
VL - 11
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 19031
ER -