How tos and FAQs

How do I prepare samples for PacBio sequencing?

DNA quality strongly affects resulting sequencing library and sequencing yield when using PacBio sequencers. It is imperative that the sample DNA is of highest quality possible and sufficient quantity since there is no amplification step during the PacBio sequencing workflow.

Here are some guidelines to follow for the best sequencing result:

  • Follow recommendations in our Sample submission guidelines.
  • DNA must be double-stranded. Single-stranded templates interfere with quantification and polymerase binding.
  • The sequencing yield is typically significantly lower for amplicons 500 - 1000 bp .
  • Samples should be eluted in Qiagen-like elution buffer (10 mM Tris-Cl, pH 8.5).
  • Try to minimize freeze and thaw cycles.
  • Do not expose samples to high temperatures (>65°C) for more than 1 hour nor to extreme pH (<6 or >9).
  • Assure that samples do not contain insoluble material and are not colored or cloudy.
  • Treat your samples with RNAse to avoid RNA in your sample.
  • Do not expose your samples to intercalating fluorescent dyes or UV radiation. Check concentration using a dedicated aliquot not to be used for sequencing.
  • Avoid in your samples: chelating agents (e.g. EDTA), detergents (like SDS, Triton-X100), denaturants (like guanidinium salts, phenol) or divalent metal cations (like Mg2+).
  • Avoid carryover contaminants from sample prep (e.g. heme, humic acids, polyphenols, etc.).
  • Do not vortex samples and if you perform AMPure bead wash pipette very gently and add some extra time for binding and elution.
  • Samples should have OD260/OD280 ratio from 1.8 to 2.0 and OD260/OD230 ratio from 2.0 to 2.2.
  • Preferably use microcentrifuge tubes.
  • Use a gel or bioanalyzer to visualize DNA quality. If sample looks degraded, re-extract or clean up using a Qiagen kit or AMPure XP beads.
  • When using any paramagnetic beads for extraction or clean up make sure there are no residual beads in the sample, these might inhibit any downstream enzymatic processes especially polymerase binding during sample preparation for sequencing.

Note: Submit also a small sample aliquot (50 ng DNA) for quality control.

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