TY - JOUR
T1 - A new approach to determining whole viral genomic sequences including termini using a single deep sequencing run
AU - Alfson, Kendra J.
AU - Beadles, Michael W.
AU - Griffiths, Anthony
PY - 2014/11/1
Y1 - 2014/11/1
N2 - Next-generation sequencing is now commonly used for a variety of applications in virology including virus discovery, investigation of quasispecies, viral evolution, metagenomics, and analyses of antiviral resistance. However, there are limitations with the current sample preparation methods used for deep sequencing of viral genomes, especially during de novo sequencing. For example, current methods are unable to capture the terminal sequences of viral genomes in an efficient and effective manner; data representing the 3' and 5' ends are typically insufficient. Methods such as Rapid Amplification of cDNA Ends address this issue but these methods can be time consuming, may require some prior knowledge of the viral sequence, and require multiple independent procedures. The current study outlines a sample preparation technique that overcomes some of these shortcomings. The method relied on random fragmentation with divalent cations and subsequent adapter ligation directly to RNA, rather than cDNA, to maximize the quality and quantity of terminal reads. The technique was tested on RNA samples from two different RNA viruses, Ebola virus and hepatitis C virus. This method permits rapid preparation of samples for deep sequencing while eliminating the use of sequence specific primers and captures the entire genome sequence, including the 5' and 3' ends. This could improve the efficiency of virus discovery projects where the terminal ends are unknown.
AB - Next-generation sequencing is now commonly used for a variety of applications in virology including virus discovery, investigation of quasispecies, viral evolution, metagenomics, and analyses of antiviral resistance. However, there are limitations with the current sample preparation methods used for deep sequencing of viral genomes, especially during de novo sequencing. For example, current methods are unable to capture the terminal sequences of viral genomes in an efficient and effective manner; data representing the 3' and 5' ends are typically insufficient. Methods such as Rapid Amplification of cDNA Ends address this issue but these methods can be time consuming, may require some prior knowledge of the viral sequence, and require multiple independent procedures. The current study outlines a sample preparation technique that overcomes some of these shortcomings. The method relied on random fragmentation with divalent cations and subsequent adapter ligation directly to RNA, rather than cDNA, to maximize the quality and quantity of terminal reads. The technique was tested on RNA samples from two different RNA viruses, Ebola virus and hepatitis C virus. This method permits rapid preparation of samples for deep sequencing while eliminating the use of sequence specific primers and captures the entire genome sequence, including the 5' and 3' ends. This could improve the efficiency of virus discovery projects where the terminal ends are unknown.
KW - Next-generation sequencing
KW - RNA virus
KW - Terminal ends
KW - Whole genome sequencing
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U2 - 10.1016/j.jviromet.2014.07.023
DO - 10.1016/j.jviromet.2014.07.023
M3 - Article
C2 - 25075935
AN - SCOPUS:84909959975
VL - 208
SP - 1
EP - 5
JO - Journal of Virological Methods
JF - Journal of Virological Methods
SN - 0166-0934
ER -