Lane mix and PhiX concentrations required for sequencing QuantSeq FWD-UMI libraries on NextSeq 500 / 550 vary slightly differ from those used for standard QuantSeq FWD libraries. For NextSeq 500 / 550 runs that consist solely of QuantSeq FWD-UMI libraries, we recommend loading 1.8 pM of the lane mix with 15 – 30 % PhiX spiked-in. For HiSeq 3000 / 4000 instruments, we recommend using 5 % PhiX spike-in (15 % spike-in may lead to PhiX over-clustering). For all other instruments, we recommend adding 15 – 30% PhiX spike-in to the lane mix and following instrument-specific loading guidelines defined by Illumina.
Loading amounts may still need to be adjusted to obtain optimal cluster densities for your respective instrument, or if sequencing chemistries are changed. Inaccuracies in library quantification can affect the loading amount required. Please ensure accurate quantification of lane mixes and lane mix dilutions when preparing for loading.
In general, we do not recommend multiplexing Lexogen libraries with libraries from other vendors in the same sequencing lane. This is related to the presence of the low-diversity 4 nt spacer immediately after the 6 nt UMI sequence. This low diversity spacer can impact read 1 quality over cycles 7 - 10 of the run. For this reason 5 – 30 % of PhiX should be spiked into all QuantSeq FWD lanemixes.
Optimal PhiX spike-in percentages also vary between instruments.
For full details please see the updated table in: Do you have recommended loading concentrations for Lexogen libraries on Illumina sequencing instruments?