This article applies to CloudbreakTM and Cloudbreak FreestyleTM sequencing runs.
Achieving optimal sequencing results depends on accurate library quantification and appropriate loading concentrations. Several factors can affect quantification accuracy and ultimately influence run density—including the chosen quantification method, library size, adapter dimers, overamplification, and variability in lab techniques.
Quantification Before Sequencing
Multiple library quantification methods are available, and certain methods may be better suited to specific library preparations. Library QC should follow the library preparation manufacturer's recommendations. A general rule for linear libraries is to qPCR quant PCR-free libraries and Qubit quant PCR-plus libraries. Libraries should be sized using a Agilent TapeStation or similar device. Note: circular Adept libraries should be sized prior to circularization and quantified using qPCR.
Library overamplification may distort the library size estimate and result in under quantification via fluorometric methods. An overamplified library is shown in the trace in Figure 1A. Avoid using an excess of PCR cycles to prevent library overamplification.
Library quantification may also be negatively impacted by the presence of adapter dimers. Adapter dimers can adversely affect sequencing by forming polonies and generating sequencing reads that do not contain a library insert. Before a sequencing run, adapter dimer may be identified as a peak around 100-150bp in the library trace. If adapter dimer is suspected prior to sequencing, an additional bead cleanup can reduce it. In Figure 1B, the blue trace illustrates a library with adapter dimer and the orange trace illustrates an identical library where an additional cleanup was performed to remove adapter dimer.
Troubleshooting After Sequencing
If a sequencing run results in a lower density than expected, first confirm the quantification of the library pool and that the recommended quantification method was used. Include a control library of known concentration if possible. Check for signs of adapter dimer or overamplification that could skew quantification results. If adapter dimer is suspected, it may also be identified after sequencing via “spikiness” in the base composition, especially in read 1 (Figure 1C). Spikes in base composition are not solely caused by adapter dimer, and knowledge of the expected base composition for the library is helpful to identify whether observed spikes are caused by adapter dimer.
The output density provided by the instrument for a sequencing run includes only those reads that have passed default quality filters. If quality is low for the run, it may be helpful to verify whether the reads are being heavily filtered due to quality, or if the total (including unfiltered) read count is also low. See the What are “pass-filter reads” and how do I calculate “pass-filter rate”? Knowledge Base article for additional information.
Loading Titration
A run that otherwise meets quality and performance expectations but is underperforming in terms of density may require higher loading. Figure 2 illustrates thumbnail images from an underloaded (A), expected (B), and overloaded (C) run. An overloaded run typically has reduced quality and reduced pass-filter reads. An underloaded run generally has good quality, but fewer polonies than desired. If titrating loading upwards maintains good quality and increases output, it is likely that the previous iteration(s) of that run were underloaded. Recommendations for sequencing run input with Cloudbreak Freestyle and Cloudbreak are included in the Cloudbreak Sequencing User Guide (MA-00058) as a starting point. Some libraries may optimally load outside the recommended input range.