meth_4.1.6 Fungal ITS gene (ITS1F and ITS4) 1-step

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

The protocol detailed here is designed to investigate the fungal diversity in environmental samples through amplification of the ITS1F and ITS4 region of the fungal ITS for paired-end sequencing on the Illumina MiSeq platform.

Primers for amplification: ITS1F and ITS4 region

ILM_ITS1F PCR primer sequence – Forward primer

Field number (space-­‐delimited), description:

  1. 5′ Illumina adapter
  2. Index (Indexed primer only)
  3. Forward primer pad
  4. Forward primer linker
  5. Forward primer (ITS1F)

Non-indexed: AATGATACGGCGACCACCGAGATCTACAC TGTCCGGCTT CG CTTGGTCATTTAGAGGAAGTAA

Indexed: AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TGTCCGGCTT CG CTTGGTCATTTAGAGGAAGTAA

ILM_ITS4 PCR primer sequence – Reverse primer

Each sequence contains different 12 base Golay barcode as described by Caporaso et al., (2012).

  1. Reverse complement of 3′ Illumina adapter
  2. Golay barcode
  3. Reverse primer pad
  4. Reverse primer linker
  5. Reverse primer (ITS4)

CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCCGTCCG GA TCCTCCGCTTATTGATATGC

Preparation of master mix for amplification ITS1F and ITS4 region (for 1 rxn)

Component

Volume (µL)

Final Conc.

10x ImmoBuffer (a)

2.50

1 X

10 mM dNTP

0.50

200 nM

50mM MgCl2

1.25

2.5 mM

ILM_ITS1F_Uv2 (10µM)

1.25

500 nM

ILM_ITS4Rv2 _XXXX (10µM)

1.25

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)

Temperature (°C)

Time (mm:ss)

Activation

94

10:00

Amplification (35 cycles)

94

00:30

55

01:00

72

01:00

Final Extension

72

10:00

Amplification method

  1. Use undiluted DNA as a first attempt, and 1:10 diluted for repeats/failed reactions. 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 ITS1F / ITS4 fragment is approximately 850 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 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 Primers

Read 1 Primer (ILM_ITS_R1v2) – for use with libraries prepared with non-indexed forward primers

Field description (space-­‐delimited):

  1. Forward primer pad
  2. Forward primer linker
  3. Forward primer

ACACTGTCCGGCTT CG CTTGGTCATTTAGAGGAAGTAA

Read 1 Primer (ILM_ITS_R1v3) – for use with libraries prepared with indexed forward primers

Field description (space-­‐delimited):

  1. Forward primer pad
  2. Forward primer linker
  3. Forward primer

TGTCCGGCTT CG CTTGGTCATTTAGAGGAAGTAA

Read 2 Primer (ILM_ITS_R2v2)

Field description (space-­‐delimited):

  1. Reverse primer pad
  2. Reverse primer linker
  3. Reverse primer

AGTCCGTCCG GA TCCTCCGCTTATTGATATGC

Index Read Primer (ILM_ITS_INDEXv3)

Field description (space-­‐delimited):

  1. Reverse complement of reverse primer
  2. Reverse complement of reverse primer linker
  3. Reverse complement of reverse primer pad

GCATATCAATAAGCGGAGGA TC CGGACGGACT