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.
• If you know how to use one of the classic methods of HMW DNA isolation, use it and avoid commercial kits. Alternatively, get inspired here.
• Use wide bore pipette tips.
• DNA must be double-stranded. Single-stranded templates interfere with quantification and polymerase binding. Treat your samples with RNAse to avoid RNA in your sample.
• Do not vortex samples even if the protocol says so, gently rock the tube instead.
• If you perform magnetic bead wash, pipette very gently and add some extra time for binding and elution. Make sure there are no residual beads in the sample, these might inhibit any downstream processes.
• Samples should be eluted in Qiagen-like elution buffer (10 mM Tris-Cl, 0,1 mM EDTA, pH 8.5). Try to minimize freeze and thaw cycles after gDNA isolation.
• Do not expose samples to intercalating fluorescent dyes, UV radiation, high temperatures (>65°C) for more than 1 hour or extreme pH (<6 or >9).
• Assure that samples do not contain insoluble material and are not colored or cloudy. Avoid carryover contaminants from sample prep (e.g. heme, humic acids, polyphenols, etc.), chelating agents (e.g. EDTA >0,1 mM), detergents (like SDS, Triton-X100), denaturants (like guanidinium salts, phenol) or divalent metal cations (like Mg²⁺).
• Samples should have OD₂₆₀/OD₂₈₀ ratio from 1.8 to 2.0 and OD₂₆₀/OD₂₃₀ ratio from 2.0 to 2.2.
• Finally, use a gel or capillary fragment analyzer (i.e. Agilent Bioanalyzer or similar) to visualize DNA quality. If sample looks degraded, re-extract or clean up.

Remarks: