Journal: Frontiers in microbiology

Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters.

doi: 10.3389/fmicb.2018.00830

Figure Lengend Snippet: FIGURE 1 | Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

Article Snippet: Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan); genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads.

Techniques: Nested PCR, Amplification, Sequencing

Journal: Frontiers in microbiology

Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters.

doi: 10.3389/fmicb.2018.00830

Figure Lengend Snippet: FIGURE 2 | Detection of serially diluted virus samples by microfluidic nested PCR followed by MiSeq amplicon sequencing. Plasmid DNA and cDNA samples containing target regions of each virus were mixed at equimolar concentrations and diluted to 104–100 copies/µL. The mixtures were subjected to the first round of MF-PCR, and the amplicon was collected and used for MFnPCR with 20 oligonucleotide primer sets for detection of 11 viruses (i.e., AiV; AsV; EV; NoV GI, GII, and GIV; HAV; HEV; RV; SaV; and HPV). CT values determined in the nested MF PCR are shown as a heat map. Amplicon size represents the size of a PCR amplicon without an Illumina tag sequence.

Article Snippet: Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan); genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads.

Techniques: Virus, Nested PCR, Amplification, Sequencing, Plasmid Preparation

Journal: Frontiers in microbiology

Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters.

doi: 10.3389/fmicb.2018.00830

Figure Lengend Snippet: FIGURE 3 | Detection and typing of viruses in samples of human feces, sewage, and contaminated oysters. The number of reads obtained by MiSeq sequencing is shown at the top of the panel, and relative abundance of the sequence reads affiliated with a specific genotype of viruses is shown in the figure. The identified genotypes are indicated in the bars.

Article Snippet: Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan); genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads.

Techniques: Sequencing