meth_4.1.9 Metagenomics Library Preparation (Australian Microbiome 2017-ongoing)

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

This protocol describes the procedure for preparing metagenomic libraries from metagenomic samples to be sequenced on the Illumina NovaSeq 6000 platform. The library preparation follows Illumina’s Illumina DNA Prep Reference Guide (Document # 1000000025416 v09, June 2020) except half-volume reactions are performed. The Illumina DNA Prep kit was previously known as the Nextera DNA Flex Library Prep kit.

Quality check of the input DNA

Verify the quality of the extracted genomic DNA using the Epoch (BioTek), 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. Use 1-15ul of DNA so that the total input amount is 0.5-250ng.
  2. DNA tagmentation follows Illumina’s Illumina DNA Prep Reference Guide (Document # 1000000025416 v09, June 2020).
  3. Library amplification follows the protocol and includes 5-12 cycles of PCR to add unique dual indexed adapters.
  4. Clean up the amplified libraries using supplied Sample Purification Beads according to the protocol.
  5. Quality check of the libraries.
  6. Verify the size of the libraries on the LabChip GX Touch HT (Perkin Elmer), LabChip GXII (Perkin Elmer) or the TapeStation (Agilent).
  7. Measure the concentration of the libraries using PicoGreen or Qubit.
  8. Pool equimolar amounts of each library and clean the pool with AMPure XP beads using a 0.8x bead:DNA ratio.
  9. Dilute the final pool to a concentration of 2-4 nM.