Library QC (LQC) control spike-in
A control library (LQC) can be added to the DNA library before clustering to increase sample diversity for difficult to
sequence low-diversity libraries. LQC should not be used if the sample library shares sequence similarities with
Lambda or
E. coli
, as distinguishing between sample reads and control library reads will be challenging.
We recommend a 5% LQC spike-in amount, however, upwards of 5% can be used if samples contain low diversity.
In cases of low diversity, we recommend using an LQC spike-in amount of approximately 20%.
Reagent and sample handling
Room temperature is defined as any temperature in the range of 18-23°C for this protocol.
All reagents and samples should be brought to room temperature and properly mixed before use. Proper mixing
includes inversion and pipette mixing when applicable. Differences in recommended mixing methods are specified
at each appropriate step.
Clustering on the cluster generator
This section describes the workflow for flow cell clustering of Onso compatible libraries in preparation for
sequencing on the Onso sequencing system. After completing this section of the guide, a clustered flow cell can
either be sequenced immediately, by following the Onso sequencing section of this guide, or can be stored at 4°C
for up to 1 week.
Each clustering reagent plate supports clustering of 2 lanes on a single flow cell.
For recommendations on sample multiplexing, refer to “General Best Practices” and “Library loading concentration
and control spike-in guidelines” sections above.
During run setup, users are able to select a sample sheet prepared and saved on a drive accessible by the PC. For
sample sheet information, templates, and guidance, refer to the PacBio document titled “Obc2fastq Reference
guide”.
Users should perform theoretical calculations with the appropriate procedure in section 3 (either 3a or
3b) to first determine the library input volume required per lane BEFORE diluting library input
concentrations to 500 pM to ensure that minimum volume requirements are met.
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