User:Jarle Pahr/16S RNA
Sequences for 16S RNA analysis:
See also http://openwetware.org/wiki/Bacterial_species_identification
Software
PHOENIX2: http://www.sciencedirect.com/science/article/pii/S0168165613002939
Primers
Weisburg et al. (1991) described several primers for broad coverage amplification. A table of primers is given below. Target-specific sequence is in upper case. Restriction site linkers (forward primers: EcoRI + SaI, reverse primers: HindIJl, BamHI, and XmaI.) are written in lower-case.
(f: forward primers, r: reverse primers)
| Name | Sequence |
|---|---|
| fDl | ccgaattcgtcgacaacAGAGTTTGATCCTGGCTCAG |
| fD2 | ccgaattcgtcgacaacAGAGTTTGATCATGGCTCAG |
| fD3 | ccgaattcgtcgacaacAGAGTTTGATCCTGGCTTAG |
| fD4 | ccgaattcgtcgacaacAGAATTTGATCTTGGTTCAG |
| rDl | cccgggatccaagcttAAGGAGGTGATCCAGCC |
| rPl | cccgggatccaagcttACGGTTACCTTGTTACGACTT |
| rP2 | cccgggatccaagcttACGGCTACCTTGTTACGACTT |
| rP3 | cccgggatccaagcttACGGATACCTTGTTACGACTT |
The primers fD1 and Fd2 above are now commonly referred to as F27, describing the sequence as AGAGTTTGATCMTGGCTCAG, using nucleotide ambiguity code where M indicates C or A. Primers rP1-rP3 differ only in nucleotide 5 of the target binding sequence (sequence excluding restriction site linker). The last 16 nucleotides (TACCTTGTTACGACTT) of primer rP1-rP3 are commonly used and referred to as 1492r. Many published primer sequences are slight variations of previously used primers.
Ambiguity codes:
M: C & A W: (A & T V: Not T Y: C & T H: Not G N: Any R: A & G
Weisburg et al. wrote that: "The primer pair designated fDl and rDl is capable of amplifying a wide variety of bacterial taxa. Replacing rDl with rPl extends the diversity of species even further, but from the perspective of amplifying the maximum number of nucleotides of 16S rDNA, rDl is preferable; it is closer to the 3' end."
For more details, see Weisburg et al. 1991.
| Name | Length (bp) | Direction | Sequence | Common name | Coverage |
|---|---|---|---|---|---|
| S-D-Bact-0008-c-S-20 | FWD | AGRGTTYGATYMTGGCTCAG | F27 | 0,0 92,6 0,0 | |
| S-D-Bact-1492-a-A-19 | REV | RGYTACCTTGTTACGACTT | 1492R | 79,3 70,6 10,2 | |
| S-D-Bact-0341-b-S-17 | CCTACGGGNGGCWGCAG | Bakt_341F | 0,5 94,9 0,0 | ||
| S-D-Bact-0785-a-A-21 | GACTACHVGGGTATCTAATCC | Bakt_805R | 90,1 90,4 0,0 | ||
| S-D-Bact-1061-a-A-17 | CRRCACGAGCTGACGAC | 1061R | 2,9 96,4 0,0 | ||
| S-D-Bact-0564-a-S-15 | FWD | AYTGGGYDTAAAGNG | 520F | 16,3 96,0 0,0 | |
| S-D-Bact-1061-a-A-17 | REV | CRRCACGAGCTGACGAC | 1061R | (A: 2.9%, B: 96.4%, E: 0.0%) | |
| S-D-Bact-0564-a-S-15 | FWD | AYTGGGYDTAAAGNG | 520F | (A: 16.3%, B: 96.0%, E: 0.0%) | |
| S-*-Univ-0515-a-S-19 | FWD | GTGCCAGCMGCCGCGGTAA | U515F | (A: 54.5%, B: 95.4%, E: 92.2%) | |
| S-D-Bact-0787-b-A-20 | REV | GGACTACHVGGGTATCTAAT | 806R | (A: 89.9%, B: 90.6%, E: 0.0%) |
Primer pairs:
S-D-Bact-0341-b-S-17/S-D-Bact-0515-a-A-19 (FWD:CCTACGGGNGGCWGCAG/REV:TTACCGCGGCTGCTGGCAC) (A: 0.0%, B: 91.2%, E: 0.0%) S-D-Bact-0564-a-S-15/S-D-Bact-0785-b-A-18. (FWD:AYTGGGYDTAAAGNG/REV:TACNVGGGTATCTAATCC)(A: 14.6%, B: 89.0%, E: 0.0%) S-D-Arch-0519-a-S-15/S-D-Bact-0785-b-A-18 (FWD:CAGCMGCCGCGGTAA/REV:TACNVGGGTATCTAATCC) (A: 88%, B: 89.1%, E: 0.7%)
S-D-Arch-0519-a-S-15/S-D-Bact-0785-a-A-21 (FWD:CAGCMGCCGCGGTAA/REV:GACTACHVGGGTATCTAATCC) (A: 86.5%, B: 87.1%, E: 0.0%).
The authors note that "reverse and forward primers with individual high coverage do not automatically qualify as an optimal primer pair."
A guideline on Universal Bacterial Identification by PCR and DNA Sequencing of 16S rRNA Gene written by Greg James is contained in PCR for Clinical Microbiology.
Klindworth et al. (2013) carried out an in silico analysis of the phylum coverage of commonly used 16S RNA amplification primers.
According to them:
"The highest overall coverage and specificity for the domain Bacteria was detected for the primers S-D-Bact-1061-a-A-17 (A: 2.9%, B: 96.4%, E: 0.0%) and S-D-Bact-0564-a-S-15 (A: 16.3%, B: 96.0%, E: 0.0%). Furthermore, 39 primers show relatively high overall coverage for more than one domain. For instance, S-*-Univ-0515-a-S-19 (A: 54.5%, B: 95.4%, E: 92.2%) detects all three domains and S-D-Bact-0787-b-A-20 (A: 89.9%, B: 90.6%, E: 0.0%) targets Bacteria and Archaea as recently reported (36)."
In the nomenclature adopted by Klindworth et al, a modified version of 27F with 4 nt listed as ambigious is referred to as S-D-Bact-0008-c-S-20 while an ambigious version of 1492R is referred to as S-D-Bact-1492-a-A-19.
See also http://en.wikipedia.org/wiki/16S_ribosomal_RNA
A workflow for 16S RNA based phylogenetic studies has been posted on the Fastgroup webpage: http://fastgroup.sdsu.edu/help.htm
According to James 2010, the following primer combinations are suitable for amplification from cultural isolates (U1+U2, U3+U4 and U5+U4) and medical specimens (U1R+U1, U3+U4 and U5+U4).
Universal 1 (U1): ACG CGT CGA CAG AGT TTG ATC CTG GCT
Universal 2 (U2): CGC GGA TCC GCT ACC TTG TTA CGA CTT
Universal 3 (U3): AGT GCC AGC AGC CGC GGT AA
Universal 4 (U4): AGG CCC GGG AAC GTA TTC AC
Universal 5 (U5): TCA AAK GAA TTG ACG GGG GC
(K=G+T)
The PCR program described by James is: "Initial denaturation at 94 C for 3 min followed by 30 cycles of 96 C for 15 s, 60 C for 1.5 min and 72 C for 2 min. A final extension step at 72 C for 5 min (...)"
According to James, a match of >98 % to an existing database sequence is generally required for identification. However, there is no universal agreement of what constitutes definitive and conclusive identification to the rank of species based on 16S RNA analysis (Janda and Abbott 2007). Recommended guidelines for use of 16S rRNA gene sequencing for microbial identification are described by Janda and Abbott (2007).
A positive (known bacterial species) and negative (no DNA) controls should be performed for very PCR.
One of the most common primer pairs for 16S RNA analysis is 27F + 1492R, amplifying nucleotides 27 to 1492 of the E. coli 16S RNA gene.
Overview of 16S RNA primers with references: http://www.lutzonilab.net/primers/page604.shtml Sequences should be checked against the original reference.
According to Frank et al. (2008), published rRNA sequences may be contain errors in the form of sequences matching the primer used for amplification when the primer sequence differs from the gene sequence:
"Our analyses of the 27f and 1492r primer-binding sites in SSU rRNA gene databases revealed that the termini of many (perhaps most) rRNA gene sequences in the databases are contaminated with sequences that do not originate from the organism that they are asserted to represent."
Bibliography
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0057923
A Quantitative Map of Nucleotide Substitution Rates in Bacterial rRNA: http://nar.oxfordjournals.org/content/24/17/3381
Review and re-analysis of domain-specific 16S primers. http://www.ncbi.nlm.nih.gov/pubmed/14607398
16S rRNA sequencing in routine bacterial identification: A 30-month experiment. http://www.sciencedirect.com/science/article/pii/S0167701206001655
Boye et al. 1999: Identification of bacteria using two degenerate 16S rDNA sequencing primers. http://www.ncbi.nlm.nih.gov/pubmed/10356793
Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, et al. (2008) Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology 74: 2461–2470. doi: 10.1128/AEM.02272-07.
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007401
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045242/
Universal Bacterial Identification by PCR
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293150/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1594676/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC391288/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC268083/
http://www.wjgnet.com/1007-9327/full/v16/i33/4135.htm
http://nar.oxfordjournals.org/content/41/1/e1.full?sid=e66b42ac-a309-47cf-8cd1-94e1229a098e#ref-13
http://www.omicsonline.com/ArchiveJCSB/2009/February/03/JCSB2.74.php
http://www.sciencedirect.com/science/article/pii/S0167701203002276
http://www.isentio.com/downloads/Isentio_Protocol_DPO_Universal_V1_V2_V3_Ver2012_1C_P1004.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1393206/
High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature
Software:
......
Sequences to order:
fd2trim: GAGTTTGATCATGGCTCAG
Removed 1bp from 5' end of Fd2 primer to better fit with criteria for LightRun sequencing primer. Hoping to be able to use the same primer both for PCR amplification and for sequencing of the PCR product.
16S RNA structure
http://delliss.people.cofc.edu/virtuallabbook/LabReadings/16s%20rDNA%20/16s%20rDNA%20SeqAnal.pdf
Conserved regions:
Hyper-variable regions:
Links
See also http://www.earthmicrobiome.org/
