GREAT AT SMALL THINGS

ITS sequencing with or without cloning

The ITS (Internal Transcribed Sequence) is a sequence located between the 16S rRNA and the 23S rRNA gene. Due to its high variability, it allows a better taxonomic resolution than the 16S rRNA gene. Therefore, we propose the sequencing of the complete ITS with the 1407F/23S30R primers. This analysis can be used as a complement to the 16S rRNA sequence to enforce the precision of the identification.

Certain cyanobacteria possess multiple copies of their 16S rRNA/ITS operon. If the direct sequencing shows that the ITS sequence is composed of multiple sequences from the different operons and is therefore unreadable, we propose to clone the ITS to separate the individual sequences. The sequencing of these isolated sequences will give single clean sequences.

For the identification based on the ITS gene sequences, we use the BLAST tool of NCBI to obtain closely related species based on their identity. Additionally, an in-depth phylogenetic study is added to complement the analysis. The phylogenetic analysis is performed with the software program MEGA7 (Kumar et al., 2016). Closely related ITS sequences are collected from Genbank (NCBI) and aligned and a phylogenetic tree will be inferred using the Neighbor-joining method.

For this analysis, a unicyanobacterial culture is required.

Literature

  • Boyer, S.L., Flechtner V.R., and Johansen J.R. (2001). Is the 16S-23S rRNA Internal Transcribed Spacer Region a Good Tool for Use in Molecular Systematics and Population Genetics? A Case Study in Cyanobacteria. Mol. Biol. Evol. 18, 1057-1069.