There are three advantages to using this method.
(1)No need to design virus-specific primers
(2)Applicable to both DNA and RNA viral genomes
(3)10 or more samples can be analyzed at a time on the iSeq100 (For genome analysis of coxsackievirus A6)
The method consists of three parts: pretreatment, random amplification, and library preparation.
The pretreatment is intended to increase the content of virus-derived nucleic acids in the sample and facilitate genome analysis. The main point of this method is to reduce host genome, ribosomal RNA, and nucleic acids derived from bacteria in advance, taking advantage of the fact that genomes in viral particles are not easily digested by Nuclease.
The random amplification using Merck millipore sigma's WTA2 kit can be used to obtain double-stranded DNA amplicon using DNA and RNA as templates. The following three points are different from the method described in the attached manual.
(1) This protocol is performed at one-fifth the scale of the protocol described in the manual.
(2) The initial denaturation temperature is changed so that DNA is also used as a template.
(3) The number of cycles of PCR amplification is increased due to the lower initial nucleic acid content.
The library preparation protocol was originally folked from "nCoV-2019 sequencing protocol for Illumina protocol V5" by Itokawa et al. Since the QIAseq FX DNA Library kit is used for library preparation in this method, multiplex analysis with the library of SARS-Cov-2 genome sequencing obtained using the protocol by Itokawa et al.