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Run Loading Cheat Sheet

The following run recommendations are for the NextSeq 500. Our NextSeq runs are set up with Basespace, but this may change in the future if it is updated to use Local Run Manager. Many of the run settings will also apply to the MiniSeq, but it uses Local Run Manager and may require different parameters. Additionally, most of these have not been run many times on the MiniSeq and may need adjustment. 10x assays have not been run on the MiniSeq yet at all, so some of the more complex recommendations may be impossible or change for that platform.

Loading concentrations may vary and are based on pools close to the desired nM concentration. For most runs, you can adjust loading concentration by pool nM concentration. For example, if you were aiming for a 4nM pool and it ends up a little higher than that, consider loading at 0.1pM lower than the recommended to accommodate. Keep in mind that the relationship of cluster density to loading concentration is not linear.

Generally, stay within loading concentrations of 1.3-1.8pM and use a default 1% PhiX spike-in. Low diversity libraries require more PhiX. The smaller the library fragment, the lower loading concentration needed, and vice versa.

Always record your run parameters, pooling, loading concentrations, and cluster density so you can gather data for future runs. Always refer to past runs when planning another run.

Run Recommendations

Prep/Run TypeLoading Concentration pM (NextSeq/MiniSeq)PhiX Spike-In %IndexesRun Parameters (Read 1 x Index 1 x Index 2 x Read 2)Notes
Stranded mRNA Prep, Ligation (Updated TruSeq)
1.6
5%
dual, 10bp "IDT-ILMN Nextera UD Index Set _ for Nextera DNA Flex" in Basespace
72x10x10x0 for 75 cycle kit, single end can run paired end, on other kits
The 5% spike-in is for the version of TruSeq updated in 2020. Older TruSeq is fine with 1%
Stranded Total RNA Prep, Ligation with Ribo-Zero Plus (Updated TruSeq)
1.6
5%
dual, 10bp "IDT-ILMN Nextera UD Index Set _ for Nextera DNA Flex" in Basespace
72x10x10x0 for 75 cycle kit, single end can run paired end, on other kits
The 5% spike-in is for the version of TruSeq updated in 2020. Older TruSeq is fine with 1%
Low input RNAseq (SMART-Seq HT PLUS Kit)
1.6-1.8 (see note)
1%
dual, 8bp "IDT-ILMN TruSeq DNA-RNA UD 96 Indexes" in Basespace
76x8x8x0 for 75 cycle kit, single end can run paired end, on other kits
RNA-seq on FFPE tissue (Takara-Clontech total RNA pico mammalian)
1.5
1%
dual, 8bp "TruSeqHT" in Basespace
76x8x8x0 for 75 cycle kit, single end can run paired end, on other kits
NexteraFLEX library prep
1.7
1%
dual, 10bp "IDT-ILMN Nextera UD Index Set _ for Nextera DNA Flex" in Basespace
72x10x10x0 for 75 cycle kit, single end can run paired end, on other kits
CRISPR Screens
1.8
20%
"CRISPR Yumi 12 index" or "Xin" but varies, ask for indexes each time, see note
varies
A few labs we work with do CRISPR screens from time to time. For each run, ask for the indexes used and check our existing Basespace files to see if they have already been used. These runs often undercluster.
Single cell RNA-seq library prep (10X) (also CITE-Seq/cell hashing/feature barcoding)
1.8
1%
single index, 8bp, 4 mixed "10X TEST" to use dummy index OR "10X Single Index" to record ONLY first barcodes/index info for 25% of run
28x8x0x91 for 150 cycle kit 28x8x0x56 for 75 cycle kit
If you choose the "10X TEST" option, your entire pool of samples will be run as one sample with dual 8bp "AAAAAAAA" indexes. If you choose the "10x Single Index" option you will need to individual enter your samples by index well, but you will only get index information for 25% of the reads.
10X MULTIOME Gene Expression
1.8
1%
dual, 10bp "10x dual 10bp Index" in Basespace for dummy index
28x10x10x90
This assay is run with a dummy index. Your entire pool of samples will be run as one sample with dual 10bp "AAAAAAAAAA" indexes.
10X ATAC-Seq
1.7
1%
standalone mode, see note
50x8x16x50 SEE NOTE
This assay has to be run in standalone mode. Before loading your run, you must change the setting. Go to Manage Instrument, then System Configuration, then Basespace Configuration, then select Standalone Instrument and Save. When loading your run, you will be asked to enter your run parameters.
10X MULTIOME ATAC
1.6
1%
custom recipe, see note
5x8x"16"x49 SEE NOTE
Due to the NextSeq 500's chemistry, this run requires a custom recipe. Recipe files for Mid and High Output runs have already been loaded on the sequencer. To use the custom recipe, put the machine in standalone mode as described above, then when asked for run parameters select the appropriate recipe from the recipe dropdown. If custom recipe files are lost or need to be re-downloaded, they can be obtained from Illumina tech support.