Input Requirements

Input Requirements

Illumina recommends 50 μl input DNA at 10 ng/μl.

Input DNA Quantitation

  • The ultimate success or failure of a library preparation strongly depends on using an accurately quantified amount of input DNA.
  • Use multiple methods of quantification to validate results.
  • Illumina recommends using fluorometric based methods for quantification, such as Qubit or PicoGreen, to provide accurate quantification for dsDNA. UV spectrophotometric based methods, such as the Nanodrop, measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides. This can give an inaccurate measurement of gDNA.
  • DNA quantification methods that rely on intercalating fluorescent dyes measure only dsDNA and are less subject to the presence of excess nucleic acids.
    • These methods require the preparation of calibration curves and are highly sensitive to pipetting error.
    • Make sure that pipettes are correctly calibrated and are not used at the volume extremes of their performance specifications.

Assessing DNA Quality

  • Genomic DNA integrity is critical for the success of TruSeq Synthetic Long-Read DNA Library Prep.
  • The DNA must be phenol free, with a size ≥ 40 kb.
  • Illumina recommends a gDNA quality check, using an agarose gel or other instrument, before proceeding with the protocol.
  • To assess quality on agarose gel, Illumina recommends running 100–500 ng of gDNA on a 0.8% Agarose gel with 200 ng of 1 kb DNA extension ladder.
  • For quality assessment guidelines, see DNA Input Recommendations in the TruSeq Synthetic Long-Read DNA Library Prep Guide.