Table 1.
The position and sequence of specific primers.
Fig 1.
Establishing the long amplicon based nanopore sequencing workflow.
A. The three pairs of primers’ location in the genome of SFTSV. B. The workflow of obtaining genome sequence of SFTSV from clinical serum specimen using nanopore sequencing technology in 10 hours. Main processes included nucleic acids extraction, genome sequence target enrichment, nanopore sequencing library preparation, real-time sequencing and analyzing.
Fig 2.
Summary of the simulated sample sequencing experiment.
A. The quantitative result of four simulated sample ranges from 25.28 to 35.61. B. Clear and precise bands showed high efficiency of the newly designed primers. Lane 1, 2, 3, 4 were M segment for S4, S3, S2, S1, whose size was nearly 3.4 kb. Lane 5, 6, 7, 8 were S segment for S4, S3, S2, S1, whose size was nearly 1.7 kb. Lane 9, 10, 11, 12 were L segment for S4, S3, S2, S1, whose size was nearly 6.4 kb. C. The number of cumulative reads increased with the sequencing time. D. The density distribution of reads quality score generated in every hour. The asterisk indicated the median quality score of reads, which decreased with time.
Fig 3.
The mapped read count and average coverage depth for every segment in the first 1 hour.
With the sequencing time passing, the mapped read count(A) and the average coverage depth(B) increased.
Fig 4.
The coverage depth for simulated samples.
An evenly distributed sequencing depth can be seen in each simulated sample’s whole genome. The figures in the first line showed the coverage depth for sequencing S1, S2, S3, S4 for 1minutes. The figures in the second, third, fourth, fifth, sixth line showed the coverage depth for sequencing S1, S2, S3, S4 for 2, 3, 4, 5, 10 minutes, respectively.
Fig 5.
Distribution of unidentical sites from Sanger sequencing.
A. A total of 10 unidentical sites between Sanger sequencing and nanopore sequencing distributed over the simulated sample whole genome. B. For M segment, 81, 408 and 2477 positions were in homopolymerize regions. For L segment, site 3089 was in the poly A region. C. Except homopolymer sites, all other unidentical sites’ second most base was identical with the Sanger sequencing.
Fig 6.
The unidentical sites distribution on the clinical serum specimen.
A. For C1 sample, 2 unidentical sites were in the poly C and poly G region. B. For C2 sample, site 408 and 2048 were in the poly T and poly C region.