Metagenomic library preparation

DNA Library Prep – Illumina DNA Prep Kit

Target insert size: ~150 bp

Final library size: ~600 bp (for 2×150 bp reads)

DNA input: 150 ng per sample

DNA Quantification & Preparation

  • Quantify DNA using Qubit dsDNA HS Assay
  • Check purity: A260/280 = 1.8–2.0
  • Dilute 150 ng DNA in 30 µL 10 mM Tris-HCl (pH 7.5–8.5)

Tagmentation

  • Prepare Tagmentation Master Mix:
    • 11 µL BLT (bead-linked transposomes)
    • 11 µL TB1 (tagmentation buffer)
  • Add 20 µL master mix to 30 µL DNA
  • Mix well, seal plate
  • Thermocycle: 55°C for 15 min, hold at 10°C

Post-Tagmentation Cleanup

  • Add 10 µL TSB (Stop Buffer), pipette to mix
  • Thermocycle: 37°C for 15 min, hold at 10°C
  • Place on magnetic stand, discard supernatant
  • Wash beads twice with 100 µL TWB
  • Leave final wash in wells (do not dry)

PCR Amplification (5 Cycles)

  • Prepare PCR Master Mix per sample:
    • 22 µL EPM
    • 22 µL Nuclease-free water
  • Add 40 µL master mix to beads
  • Add 10 µL pre-paired index adapters (i5+i7)
  • Thermocycle:
    • 68°C 3 min
    • 98°C 3 min
    • 5 cycles: 98°C 45s, 62°C 30s, 68°C 2 min
    • 68°C 1 min → Hold at 10°C

Library Cleanup (Ampure beads)

  • Transfer 45 µL PCR product to new plate
  • Add 40 µL water + 45 µL SPB → mix, incubate 5 min
  • Transfer 125 µL to new plate with 15 µL SPB → mix, incubate 5 min
  • Wash twice with 200 µL 80% EtOH
  • Air dry beads for 5 min
  • Elute in 32 µL RSB, transfer 30 µL clean library

Library QC & Pooling

  • Tapestation for analyzing the size of library
  • Qubit for quantification of the final pooled library.