Oxford nanopore technology does not experience any fragment size limitation, not even size bias in the sense of preferential loading of short fragments or similar. On the other hand, it is very sensitive to any kind of inhibitors. Inhibition of nanopore activity might significantly decrease sequencing output. So carefully check samples for presence of inhibitors.

If you are not able to get rid of certain amount of inhibitors, we can construct low input library using few PCR cycles, inhibitors would be diluted and additionally purified. We recommend you freeze samples directly after isolation and quality control and avoid repeated thaw/freeze cycles.

Note: For metagenomic analyzes based on the sequencing of long amplicons (e.g. 16S), gDNA can be prepared in a conventional manner as in the case of Illumina technology. It is important that the DNA is free of inhibitors of the PCR reaction.

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: