What’s the best loading concentration for my library? Is my run overloaded? Underloaded?
The optimal loading concentration will depend on library size, library quality, and, in some cases, the application for each run. Use the recommendations in the Cloudbreak™ Sequencing User Guide as a starting point to determine your optimal loading concentration, then adjust based on run performance. A run is generally considered overloaded if the pass filter (PF) rate is under 90% and the thumbnail shows significantly more polonies than what the final run output reports. Underloading results in sparse thumbnails and quality metrics that remain high.
All dimers and high molecular weight products must be removed from the library pool before dilution and denaturation. Pooled libraries should contain similar size distributions since loading concentration is based on a particular size range. Performance can be impacted if libraries with disparate size ranges are pooled together.
Multiple library quantification methods are available, and certain methods may be better suited to specific library preparations. Follow the library preparation manufacturer's recommendations, and avoid overamplification or excessive adapter dimer, which may skew quantification. For more help with library quantification, see What are some best practices for library quantification prior to sequencing?
You can load the same library at different concentrations on the same flow cell with individually addressable lanes (IAL). Load one lane at a higher concentration than the other and see which concentration provides the best output.
If you are running a counting application on AVITI™, Expert Mode HD can enable an increase in run throughput with a minor quality impact (slightly reduced Q30).
Before starting your sequencing run, ensure that you are using the correct sequencing recipe for your given library size, as this can affect sequencing output. For libraries with an average size < 300 bp, we recommend using the Short Insert Recipe. For libraries with an average size > 750 bp, we recommend using the Long Insert Recipe. See Which sequencing recipe should I use for my library insert size? for more details.
Check the thumbnail. Compare your run to the reference images in Figure 1 of an optimal, overloaded, and underloaded sequencing run. If your thumbnail looks blurry or obstructed, contact Element Biosciences Support.
Figure 1. Example thumbnail images for sequencing runs: (A) Underloaded run, (B) Optimal run, and (C) Overloaded run. The image on the left in each group is the full view and the right is zoomed in at 350%.
Review sequencing run metrics like quality and percent passing filter (PF). Low output despite high Q30 and high PF, with a sparse thumbnail image (Figure 1A) indicates underloading. Low Q30 and a pass filter rate (PF) below ~90% can indicate overloading and will be accompanied by a crowded thumbnail image (Figure 1C). For more information on PF, see What are pass-filter reads?