Sequencing of Open-ST library¶
Quantification¶
We recommend quantifying your libraries for sequencing using the KAPA Library Quantification Kit.
Loading and sequencing¶
The optimal loading concentration depends on the sequencer used. For the Illumina® NovaSeq 6000 we obtained optimal clustering at a loading concentration of 130 pM. For the Illumina® NextSeq 2000 sequencing system we recommend a loading concentration of 650 pM.
Moreover, Illumina suggests a minimum spike-in of 1% PhiX into the pool as a quality control for cluster generation, sequencing, and alignment.
In our setup, the following read lengths were used:
| Read | Cycles |
|---|---|
| Read 1 | 28-32 |
| Index 1 | 8 |
| Index 2 | NA |
| Read2 | 90+ |
Expected (data) output¶
Either when using your own sequencing equipment or relying on a sequencing facility, you will get access
to (most likely) already demultiplexed
fastq files; otherwise, you can get access to the raw basecall files in bcl format.
Either of these files shall be used as the input for spacemake later.