Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome.
The recent development and advancement of next-generation sequencing (NGS) technologies have enabled the characterization of the human genome at extremely high resolution. In the retrovirology field, NGS technologies have been applied to integration-site analysis and deep sequencing of viral genomes in combination with PCR amplification using virus-specific primers. However, virus-specific primers are not available for some epigenetic analyses, like chromatin immunoprecipitation sequencing (ChIP-seq) assays. Viral sequences are poorly detected without specific PCR amplification because proviral DNA is very scarce compared to human genomic DNA. Here, we have developed and evaluated the use of biotinylated DNA probes for the capture of viral genetic fragments from a library prepared for NGS. Our results demonstrated that viral sequence detection was hundreds or thousands of times more sensitive after enrichment, enabling us to reduce the economic burden that arises when attempting to analyze the epigenetic landscape of proviruses by NGS. In addition, the method is versatile enough to analyze proviruses that have mismatches compared to the DNA probes. Taken together, we propose that this approach is a powerful tool to clarify the mechanisms of transcriptional and epigenetic regulation of retroviral proviruses that have, until now, remained elusive.
Center for AIDS Research, Kumamoto University, Japan.|International Research Center for Medical Sciences, Kumamoto University, Japan.|Priority Organization for Innovation and Excellence, Kumamoto University, Japan.
Paola
P
Miyazato
Center for AIDS Research, Kumamoto University, Japan.|International Research Center for Medical Sciences, Kumamoto University, Japan.|Priority Organization for Innovation and Excellence, Kumamoto University, Japan.
Hiroo
H
Katsuya
Center for AIDS Research, Kumamoto University, Japan.|International Research Center for Medical Sciences, Kumamoto University, Japan.|Priority Organization for Innovation and Excellence, Kumamoto University, Japan.
Asami
A
Fukuda
Department of Medical Physics, Faculty of Life Sciences, Kumamoto University, Japan.
Yoshikazu
Y
Uchiyama
Center for AIDS Research, Kumamoto University, Japan.|International Research Center for Medical Sciences, Kumamoto University, Japan.|Priority Organization for Innovation and Excellence, Kumamoto University, Japan.
Misaki
M
Matsuo
Center for AIDS Research, Kumamoto University, Japan.|International Research Center for Medical Sciences, Kumamoto University, Japan.|Priority Organization for Innovation and Excellence, Kumamoto University, Japan.
Michiyo
M
Tokunaga
Department of Medical Cell Biology, Institute for Molecular Biology and Embryology, Kumamoto University, Japan.
Shinjiro
S
Hino
Department of Medical Cell Biology, Institute for Molecular Biology and Embryology, Kumamoto University, Japan.|Core Research for Evolutionary Science and Technology (CREST), Japan Science of Technology Agency, Tokyo, Japan.
Mitsuyoshi
M
Nakao
Center for AIDS Research, Kumamoto University, Japan.|International Research Center for Medical Sciences, Kumamoto University, Japan.|Priority Organization for Innovation and Excellence, Kumamoto University, Japan.
Yorifumi
Y
Satou
D002460:Cell Line; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D011533:Proviruses / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D016662:Virus Integration