meth_4.1.8 Illumina Nextera XT Library Preparation for Metagenomics (Marine Microbes Project 2015-2017)

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

This protocol describes the procedure for preparing metagenomic libraries from marine samples to be sequenced on the Illumina HiSeq 2500 platform. The library preparation follows Illumina’s Nextera XT DNA Library Prep Reference Guide (Document # 15031942 v01, January 2016) except for the bead-based normalisation and pooling steps.

Quality check of the input DNA

Verify the quality of the extracted genomic DNA using the NanoDrop (Thermo Scientific) or DropSense 16 (Trinean, formerly known as Xpose). The concentration should be verified with the Qubit or PicoGreen assay (Invitrogen).

Library preparation

  1. Dilute 1 ng of DNA to a concentration of 0.2 ng/μL.
  2. DNA tagmentation follows Illumina’s Nextera XT DNA Library Prep Reference Guide (Document # 15031942 v01, January 2016).
  3. Library amplification follows the protocol and includes 12 cycles of PCR to add indexed adapters.
  4. Clean up the amplified libraries using AMPure XP or AxyPrep Mag PCR clean-up beads according to the protocol.

Quality check of the libraries

  1. Verify the size of the libraries on the LabChip GXII (Perkin Elmer) or the TapeStation (Agilent).
  2. Measure the concentration of the libraries using Qubit, PicoGreen, or qPCR.
  3. Normalise libraries and pool manually, do not follow bead-based normalisation and pooling protocol detailed in the sections of the Prep Guide entitled “Normalize Libraries” and “Pool Libraries”.
  4. Dilute the final pool to a concentration of 2 nM.