meth_4.1.1 Bacterial 16S rRNA gene (27F and 519R) 1-step using Immolase™ DNA Polymerase (Bioline)

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 (single and 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

  1. 5′ Illumina adapter
  2. Index (indexed primer only)
  3. Forward primer pad
  4. Forward primer linker
  5. Forward primer (1391f)

Non-indexed: AATGATACGGCGACCACCGAGATCTACAC TATGGCGAGT GA AGAGTTTGATCMTGGCTCAG

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)

Final concentration

10x ImmoBuffer (a)

2.50

1 X

10 mM dNTP

0.50

200 nM

50mM MgCl2

1.25

2.5 mM

ILM_27F_Uv3 (forward) (5 µM)

2.50

500 nM

ILM_519R_XXXX (5µM)

2.50

500 nM

Immolase DNA Polymerase (5U/µL)(a)

0.20

1 Unit

H2O

14.55

-­‐

Template

1.0

-­‐

Total Volume

25

-­‐

a        IMMOLASE™ DNA Polymerase (Bioline, #BIO-­‐21047)

Thermocycler conditions for amplification (for 96 well thermocycler)

Reaction Step

Temperature (°C)

Time (mm:ss)

Activation

95

10:00

Amplification (35 cycles)

94

00:30

55

00:10

72

00:45

Final Extension

72

10:00

Amplification method

  1. 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.
  2. Amplify samples with conditions outlined above
  3. Run amplicons on an agarose gel. Expected band size for 27F/519R is approx. 530 bp.
  4. Clean and normalise samples in a one-step process using the SequalPrep Normalisation Plate Kit according to manufacturer instructions (Invitrogen Cat No. A10510-01
  5. Combine equivalent volumes of normalised amplification into a single maximum-recovery tube.
  6. Perform a double cleanup of the pool using AMPure XP or AxyPrep Mag PCR clean-up beads.
  7. Perform library QC on the pool using Qubit (concentration) and TapeStation (size). Calculate final molarity of the pool.
  8. 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