Safety guidelines
Please contact the Environmental Health and Safety (EHS) Manager to understand appropriate disposal of
reagents and materials used in this operations guide as guidelines may differ per user site.
Refer to SDS (102-855-900) and hazard insert (103-033-900) for reagent specific hazard ratings for the Onso
clustering reagent plate.
Refer to SDS (102-850-900) and hazard insert (103-032-600 and 103-032-800) for reagent specific hazard ratings
for the sequencing reagent pack.
For all other reagents, refer to the Safety Data Sheet (SDS) for information on reagent hazards and protocols for
safe handling, use, storage, and disposal.
General best practices
DNA library input
DNA libraries should contain Onso indexed adapters.
DNA libraries should be accurately quantitated with the Onso Library quant kit (see Procedure & checklist - qPCR
Quantification of Onso libraries) or a comparable qPCR quant kit using appropriate primers. There should be at
least 16 µL of starting dsDNA library available for use at a concentration of ≥500 pM per lane of a flow cell before
clustering.
Multiplexing
When multiplexing samples, a minimum of 4 samples is advised for pooling prior to clustering if users do not
choose to use the Library QC (LQC) spike-in. Using fewer than 4 samples in the absence of an LQC spike-in will
result in too low sample diversity during sequencing.
If multiplexing samples in the absence of LQC spike-in, a minimum of 8 Onso indexed adapters are required to
supply sufficient index diversity.
For recommendations around appropriate LQC spike-in amounts for multiplexing purposes, refer to the section
below titled “Library loading concentration and control spike-in guidelines”.
Library loading concentration and control spike-in guidelines
Optimal loading concentration
Determining the best loading concentration to achieve optimal cluster density will depend upon several factors,
such as the starting material of your library, average library size, and library prep method. Over-clustering will
compromise overall sequencing run performance, whereas under-clustering will not maximize potential data
output. Therefore, striking a balance between over-clustering and under-clustering will increase the likelihood of
obtaining optimal sequencing data quality, while reducing the amount of sequencing that may be required to
achieve target Gb or fold coverage.
To determine the optimal loading concentration for your library, performing a library input titration is advised for
each new library type being clustered.
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