PMC4847596_01
Base-calling electropherogram of borrelial 16S rRNA gene sequencing, using M2 as the sequencing primer.. Notes: Computer-generated electropherogram is presented. BLAST alignment algorithm showed that the first unambiguous 110 bases, TTAACCCAACACCTCACAGCACGAGCTGACGACAACCATGCAGCACCTGTATATAGACCCCAAACGGGGAATAATTATCTCTAACTATATCCTATATATGTCAAGCCCTG, represent a signature sequence of the Borrelia burgdorferi sensu lato 16S rRNA gene (GenBank sequence ID CP009656, range: 445,000-445,109). Visual analysis showed that the immediately downstream 100-base segment with numerous double peaks is a composite of two superimposed sequences shown later: A:GTAAGGTTCCTCGCGTATCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCACTCTTGCGAGCATACTCCCC; and B:TAAGGTTCCTAGCGTATCATCGAATTAAACCACATTCTCCACCGCTTGTGGGGGCCCCCGTCAATTCCTTTGAGTTTCACTCTTGCGAGCATACTCCCCA.. Abbreviations: BLAST, Basic Local Alignment Search Tool; rRNA, ribosomal RNA.
imcrj-9-101Fig1.jpg
PMC4847596_01_imcrj-9-101Fig1.jpg
f1-imcrj-9-101
The ambiguous DNA sequence is obviously caused by a shift in the reading frame (Figure 1). B. burgdorferi 16S rRNA gene sequence ID CP009656, there is a shift in the reading frame in Figure 1, caused by a sequence B superimposed on a sequence A. Based on known sequences retrieved from the National Center for Biotechnology Information database, sequence B has a maximum 97% ID match with a highly conserved 16S rRNA gene segment commonly shared by numerous spirochete species, including B. burgdorferi (ID CP009656, range: 445,111-445,210), B. turicatae (ID KP861623, range: 237-336), B. afzelii (ID CP009058, range: 447,714-447,813), B. hermsii (ID CP011060, range: 447,836-447,935), and Treponema pallidum (ID CP007548, range: 232,098-232,197).
BLAST alignment report showing sequence B in the search query with a maximum 97% ID match with a common spirochete 16S rRNA gene sequence cataloged in the GenBank.. Abbreviations: BLAST, Basic Local Alignment Search Tool; rRNA, ribosomal RNA.
imcrj-9-101Fig2.jpg
PMC4847596_01_imcrj-9-101Fig2.jpg
f2-imcrj-9-101
One of these examples was copied from the BLAST report returned from the GenBank and is illustrated in Figure 2. However, alignment of the 210-base sequence composed of the 110-base unambiguous sequence and the 100-base sequence B against the sequence ID CP009656.1 showed deletion of one "G" nucleotide at reference position 445,110 and three single-base substitutions: one C A at reference position 445,121; one G T at position 445,146; and one C G at position 445,161 (Figure 2).