Why am I seeing low sequencing output even though I’m using the same loading concentration for this same library as before?
Unexpected low output from a previously optimized library is usually caused by a change in library quality or a mistake in library preparation. Common contributors include improper denaturation, dilution or quantification errors, DNA loss from non–low-bind plasticware, degraded library, upgraded AVITI™ system, or compromised reagents.
Excess exposure to sodium hydroxide (NaOH) or delays in neutralization reduce amplification efficiency. Always use fresh reagents and load immediately after denaturation. Libraries should not remain in NaOH for more than 5 minutes before neutralization with an equal volume of Tris. When preparing multiple samples, avoid over-denaturing early samples while finishing later ones by letting them sit in the NaOH for too long.
Inaccuracies in quantification or dilution can lead to underloading despite an unchanged workflow. Re-quantify both the stock library and the 1 nM intermediate using multiple instruments, if applicable, to confirm consistency. Pipetting variability increases at low volumes and concentrations, and insufficient mixing can further impact accuracy. Confirm pipette calibration and ensure thorough mixing at each step.
Material loss to standard plastic surfaces during library preparation should be considered. Use low-bind plasticware for all DNA handling steps to reduce variability and minimize loss.
Degradation due to poor storage conditions, repeated freeze-thaw cycles, or inherently short fragments can reduce output. Inspect the library trace for shifts in fragment distribution. A broadened peak or the presence of smaller fragments can indicate degradation. Compare against traces from prior successful runs to assess consistency.
With an AVITI24™, loading concentrations may need to be adjusted depending on the sequencing kit used and sequencing yield currently obtained on the AVITI. Find more information here.
NaOH and Tris used during denaturation should be prepared fresh for each run. Verify that all reagents are not past their expiration date. While the AVITI systems allow runs to proceed with expired sequencing kits, performance cannot be guaranteed. Improper thawing will also impact run performance. Make sure that cartridges are thawed to our recommendations in the Cloudbreak Sequencing User Guide.