Fig 1.
Double peaks from Sanger sequencing electropherogram of the 16S rRNA-gene PCR product of strain GS2.
(A) shows the actual PCR product sequence and location of variants in the electropherogram, where double peaks are found, and (B) shows the actual sequence vs. expected sequence of the variants where the actual sequence is the sequence obtained from Sanger sequencing and the expected sequence is the sequence derived from the alignment of the 8 copies of the 16S rRNA genes of GS2. The red arrow indicates the base where sequencing machine showed a deviation from the 16S rRNA sequence of GS2. Nucleotides were color-coded based on the electropherogram result; G (black), T (red), A (green), and C (blue) and nearest locations where the variations are found in the 16S rRNA genes are placed above the electropherogram.
Fig 2.
Representation of the number of 16S rRNA copies in GS2.
Whole genome sequencing data is shown in (A) (Ortho 1–Ortho 8), and cloned 16S rRNA genes (Clone A–Clone F) with their variations are shown in (B). Nucleotides were colored based on their color-coded based from the electropherogram result; G (black), T (red), A (green), and C (blue) and nearest locations where the variations are found in the 16S rRNA genes are placed above the schematic. The purple arrows show where the region of the Taq polymerase error produced variations.
Fig 3.
Effect of the amount of PCR template and ratio on the electropherogram and sequencing results.
A, B, and C correspond to 5:5 ratio of clones F and A with 1, 10, and 100 ng of PCR template concentration while D, E, and F correspond to 8:2 ratio of clones F and A having 1, 10, and 100 ng of PCR templates, respectively. The electropherograms showed are based from the 400–420 bp region of the 16S rRNA gene Sanger sequencing which showed multiple variations between the Clones F and A.
Fig 4.
Phylogenetic tree of 16S rRNA genes from WGS data of S. fonticola GS2 with the cloned PCR products, actual PCR product, and the sequence that mimics the maximum variation.
Copies of the 16S rRNA genes from WGS (Ortho1-8), cloned PCR products (Clone A-F), actual PCR product, and a mimic of the sequence with the highest variation, were compared with other species from the genus Serratia using a phylogenetic tree. Bootstrap values obtained with 1000 repetitions are indicated as percentages at all branches. The scale bar represents an evolutionary distance of 0.0020. GenBank accession numbers of the different Serratia species are provided in parentheses.
Table 1.
Summary of analyses done on whole genome sequences involving 1616 genomes in different phyla that exclusively used pacific biosciences and oxford nanopore technology sequencing platforms.