meth_4.1.2 Bacterial 16S rRNA gene (27F and 519R) 1-step using KAPA HiFi Hot Start ReadyMix (Roche)
Contributor(s): Ramaciotti Centre for Genomics, UNSW, Sydney
The bacterial communities in environmental samples are investigated by amplification of the 16S rRNA gene using the primer set 27F and 519R (dual indexed) and paired-end sequencing on the Illumina MiSeq platform.
Primers for amplification: 27F (Lane 1991) and 519R (Lane et al., 1993)
ILM_27F_Uv3 –forward primer
Both un-indexed and indexed versions are used, depending on the need for dual indexing
- 5′ Illumina adapter
- Index
- Forward primer pad
- Forward primer linker
- Forward primer (1391f)
Indexed: AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TATGGCGAGT GA AGAGTTTGATCMTGGCTCAG
ILM_519R_NNNN – reverse primer
- Reverse complement of 3′ Illumina adapter
- Golay barcode*
- Reverse primer pad
- Reverse primer linker
- Reverse primer (EukBr)
CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCAGTCAG GG GWATTACCGCGGCKGCTG
* This primer includes a 12 base Golay barcode as described by Caporaso et al., (2012)
Preparation of master mix for amplification (for 1 rxn)
|
Component |
Volume (µL) |
|
KAPA HiFi Hot Start Readymix (2x) (a) |
10.0 |
|
H2O |
12.0 |
|
Forward primer ILM_27F_Uv3 (10 µM) |
1.0 |
|
Reverse primer ILM_519R_XXXX (10 µM) |
1.0 |
|
Template |
1.0 |
|
Total Volume |
25 |
(a) Kit code KK2601 or KK2602
Thermocycler conditions for amplification (for 96 well thermocycler)
|
Reaction Step |
Temperature (°C) |
Time (mm:ss) |
|
Activation |
95 |
3:00 |
|
Amplification (35 cycles) |
98 |
00:20 |
|
55 |
00:10 |
|
|
72 |
00:45 |
|
|
Final Extension |
72 |
5:00 |
Amplification method
- Use neat DNA for initial attempt, 1:10 dilution for failed samples (2nd attempt). Please note that for the BASE project 1:10 was the default for the initial attempt.
- Amplify samples with conditions outlined above.
- Run amplicons on an agarose gel. Expected band size for 27F/519R is approx. 530 bp.
- Clean and normalise samples in a one-step process using the SequalPrep Normalisation Plate Kit according to manufacturer instructions (Invitrogen Cat No. A10510-01).
- Combine equivalent volumes of normalised amplification into a single maximum-recovery tube.
- Perform a double cleanup of the pool using AMPure XP beads.
- Perform library QC on the pool using Qubit (concentration) and TapeStation (size). Calculate final molarity of the pool.
- Dilute pool to 4 nM.
Sequencing of the 27F and 519R fragment
- Sequencing Primers Read 1 Primer: ACACTATGGCGAGTGAAGAGTTTGATCMTGGCTCAG
- Read 2 Primer: AGTCAGTCAGGGGWATTACCGCGGCKGCTG
- Index Primer: CAGCMGCCGCGGTAATWCCCCTGACTGACT