meth_4.1.7 Illumina TruSeq Nano Library Preparation for Metagenomics (BASE)

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

This protocol describes the procedure for preparing libraries from metagenomic samples to be sequenced on the Illumina HiSeq 2500 platform. The library preparation follows Illuminas TruSeq Nano DNA Sample Preparation Guide (Part # 15041110 Rev. B, November 2013).

Quality check of the input DNA

Verify the quality of the extracted genomic DNA using the Caliper LabChip GX (Perkin Elmer) or the TapeStation genomic DNA assay (Agilent), and the nanodrop (Thermo Scientific) or Xpose (Agilent). The concentration should be verified with the Qubit or PicoGreen assay (Invitrogen).

Library Preparation

  1. Dilute 200 ng of DNA in a volume of 52.5 μL of RSB or EB.
  2. Shear the DNA as per protocol following the 550 bp Covaris settings.
  3. Perform clean-up of the DNA using AMPure XP beads following the 550 bp insert bead ratio. End-repair, A-tailing and ligation of adapters follow the HT Nano Protocol of the TruSeq Nano DNA Sample Preparation Guide (Part # 15041110 Rev. B, November 2013). Use the DAP plate to index each individual library.
  4. Follow the protocol to perform the PCR, using 8 PCR cycles.
  5. Clean-up the PCR-amplified DNA following the 550 bp insert ratio of AMPure XP beads.

Quality check of the libraries

  1. Verify the size of the libraries on the LabChip GX HS DNA chip, the Bioanalyzer HS or the TapeStation.
  2. Measure the concentration of the libraries using Qubit HS or picogreen.
  3. Dilute the libraries to 10 nM stocks and pool an equal amount of each library. Bring the final 10 nM pool to 2nM.