meth_4.1.4 Eukaryotic 18S v4 rRNA gene (TAReuk454FWD1 and modified TAReukREV3) 1-step

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

The protocol detailed here is designed to amplify the V4 region of the 18S rRNA gene for paired-end 18S community sequencing on the Illumina MiSeq platform. This protocol is based on Illumina’s 16S Metagenomic Sequencing Library Preparation guide and the protocol used by Ocean Sampling Day, modified to amplify the target and add indexed adapter sequences in a single PCR step.

Primers for amplification: V4 region

A full list of primer sequences can be requested from the sequencing facility.

Forward primer

Field number (space-delimited), description:

  1. 5′ Illumina adapter
  2. Nextera XT i5 index sequence
  3. Illumina forward overhang sequence
  4. 18S V4 forward

AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CCAGCASCYGCGGTAATTCC

Reverse primer

Field number (space-delimited), description:

  1. Reverse complement of 3′ Illumina adapter
  2. Nextera XT i7 index sequence
  3. Illumina reverse overhang sequence 18S V4 reverse

CAAGCAGAAGACGGCATACGAGAT XXXXXXXX GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG ACTTTCGTTCTTGATYRATGA

Preparation of master mix for amplification V4 region (for 1 rxn)

Component

Volume (µL)

KAPA HiFi Hot Start Readymix (2x) (a)

12.5

H2O

9.0

Forward primer (10 µM)

1.25

Reverse primer (10 µM)

1.25

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

98

0:30

Amplification (10 cycles)

98

0:10

44

0:30

72

0:15

Amplification (20 cycles)

98

0:10

62

0:30

72

0:15

Final Extension

72

7:00

Amplification method

  1. Use undiluted DNA as a first attempt, and 1:10 diluted for repeats/failed reactions
  2. Amplify samples with conditions outlined above.
  3. Run amplicons on an agarose gel. Expected band size for 18S V4 is approximately 536 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.

Resources

16S Metagenomic Sequencing Library Preparation (Illumina Part # 15044223 Rev. B) available here: http://www.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentati on/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf

LifeWatch Italy Ocean Sampling Day 2014 Protocol – available here: http://mb3is.megx.net/osd- files/download?path=/2014/protocols&files=OSD2014_protocol_B_18S_V4andV9_Sequencing_LifeWath_MoBiLab_BARI.pdf