meth_4.1.3 Archaeal 16S rRNA gene (A2F and 519R*) 2-step using Immolase™ DNA Polymerase (Bioline)

Contributor(s): Ramaciotti Centre for Genomics, UNSW, Sydney

The archaeal communities in environmental samples are investigated by amplification of the archaeal 16S rRNA gene (A16S) and paired-end sequencing on the Illumina MiSeq platform. The preparation of archaeal 16S enriched libraries uses a two-stage PCR strategy. The first round of PCR uses locus specific primers with overhang adapters (A2F_Nex and 519R_Nex). The locus specific region of the forward primer was based on the A2F primer from Reysenbach et al., (1995), which is specific to archaeal targets. The reverse primer has a locus specific region that is universal for prokaryotes. The second round PCR and subsequent steps of library preparation and sequencing follow the “16S Metagenomic Sequencing Library Preparation” guidelines from Illumina.

Primers for amplification: A2F and based on 519R*

A2F_Nex PCR Primer Sequence – Forward primer

  1. Forward overhang
  2. Locus specific sequence (A2F primer)

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG TTCCGGTTGATCCYGCCGGA

519R_Nex – Reverse primer*

  1. Reverse overhang
  2. Locus specific sequence (16S universal primer)

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GWATTACCGCGGCKGCT

*PLEASE NOTE THAT THE FINAL BASE (G) ON THE 519R PRIMER IS MISSING. THIS IS CONSISTENT ACROSS A16S AMPLICON DATA GENERATED.

 

Preparation of master mix for amplification A2F / 519R* (for 1 rxn)

Component

Volume (µL)

PCR Grade H2O(a)

17.55

10x immoBuffer(b)

2.50

50 mM MgCl2(b)

0.75

10 mM dNTPs

0.50

A2F Nex primer (10 µM)(c)

1.25

519R Nex primer (10 µM) (c)

1.25

Immolase DNA polymerase(b)

0.20

Template DNA

1.0

Total Volume

25

(a)     PCR grade water was purchased from MoBio Laboratories (MoBio Labs: Item#17000l 11)

(b)     IMMOLASE DNA Polymerase Kit (Cat number: BIO-2146)

(c)     Final primer concentration in reaction: 0.5 µM

Thermocycler conditions for amplification (for 96 well thermocycler)

Reaction Step

Temperature (°C)

Time (mm:ss)

Activation

95

10:00

Amplification (35 cycles)

95

00:30

60

00:15

72

00:50

Final Extension

72

5:00

Amplification method

  1. Use undiluted DNA for first attempt, and 1:10 dilution for second attempt (if failed samples). 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. The expected band size is approximately 520 bp.
  4. If there is no band present, repeat PCR using a 1:10 dilution of the sample. Use the concentration of the DNA extract to determine if the DNA should be further diluted or used at higher volumes.
  5. Clean amplicons with Agencourt AMPure XP beads, according to manufacturer’s instructions.
  6. Perform a second round PCR (Index PCR) following Illumina’s 16S Metagenomic Sequencing Library Preparation, section Index PCR (part #15044223). A half reaction (25 µL total reaction volume) can be used.
  7. Run Index PCR products on an agarose gel. The expected band size is approximately 630bp.
  8. Clean and normalise samples in a one-step process using the SequalPrep Normalisation Plate Kit according to manufacturer instructions (Invitrogen Cat No. A10510-01).
  9. Combine equivalent volumes of normalised amplification into a single maximum-recovery tube.
  10. Perform a double cleanup of the pool using AMPure XP beads.
  11. Perform library QC on the pool using Qubit (concentration) and TapeStation (size). Calculate final molarity of the pool.
  12. Dilute pool to 4 nM.