How tos and FAQs

Additional sample submission guidelines - 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. Treat your samples with RNAse to avoid RNA in your sample.
  • Do not vortex samples and 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:

gDNA for assemblies should be of high quality. Otherwise there is a risk of sense/antisense chimeras and big chance of highly non-uniform coverage of genome (incomplete assembly).

Pictured is an example of a high quality gDNA (lanes 1 and 2) obtained by using MagAttract HMW DNA kit (Qiagen). It is generally recommended to use minimal amount of starting material, check the quality of isolate immediately, make aliquots and freeze/fridge them straight away.

  • Long-term storage: -20°C, >20 ng/μl
  • Short-term storage: 4°C, >20 ng/μl

 

Amplicons should be rather longer than shorter. Design your amplicons >1000 bp, if possible. Otherwise there is a risk of unbalanced loading and also the loading efficiency may decrease. The sequencing yield is typically significantly lower for amplicons <1000 bp!

 

 

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