Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO. Nucleic acids research. 2013. Pubmed
Glossary Name (Alm et al., 1996). Probe designation according to Alm, E. W., Oerther, D. B., Larsen, N., Stahl, D. A., Raskin, L. (1996). The oligonucleotide probe database. Appl Environ Microbiol 62: 3557-9. Abstract (PUBMED). Position. Probe position according to the E. coli gene numbering. Sequence. Sequence in IUPAC code: R=G/A, Y=T/C, M=A/C, K=G/T, S=G/C, W=A/T, H=A/C/T, B=G/T/C, V=G/C/A, D=G/A/T, N=G/A/T/C Tm. Dissoziation temperature according to: Tm=64.9 + 41 x ((G + C - 16.4)/length). Hybridization efficiency. Use this tool to assess in silico sensitivity (i.e. the hybridization efficiency of the oligonucleotide with its fully complementary target sequence, calculated with ProbeMelt. Formamide. Percent formamide in the hybridization buffer for optimal hybridization conditions in FISH experiments. Coverage. Coverage of the three domains calculated using the SILVA reference database 106 if no or a single mismatch is allowed. The detailed method is described in Klindworth et al., 2012. Nucleic Acids Res. 10.1093/nar/gks808 Full Text Check specificity/coverage. Use these options to reveal the in silico specificity (i.e. number of matching rRNA sequences outside the target taxon) and coverage (i.e. percentage of matching rRNA sequences within the target taxon) of an oligonucleotide against the most recent SSU and LSU rRNA sequence databases.
