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Sequencing frequently asked questions for the Illumina miRNA prep kit

What is the recommended sequencing read length?

  • 1 x 72 single read length with dual 10 bp indexes.

  • Note: the sequencing of UMIs is required for the Illumina DRAGEN miRNA analysis pipeline, customers must sequence 1 x 72 bp in order to capture the UMI sequence.

How much PhiX needed in the sequencing run?

  • PhiX is not required, though 1-2% can be added to the run for additional sequencing metrics, such as error rate.

How many reads per sample are recommended?

  • Illumina recommends beginning with 5 - 10 million reads per sample. This number may need to be optimized based on the customer’s experimental aim and sample type.

On what instruments has the Illumina miRNA Prep library been tested?

  • iSeq 100

  • MiSeq

  • MiniSeq

  • NextSeq 500/550

  • NextSeq 1000/2000

  • NovaSeq 6000

What is the recommended loading concentration for each Illumina sequencer?

  • This information can be found in the library prep Product Documentation:

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Is it okay to sequence libraries with some adapter dimer, even on patterned flow cells?

  • Yes. The miRNA libraries and adapter dimer are very similar in size, so adapter dimer does not “overtake the run.” Therefore, there will still be usable miRNA reads even in libraries with adapter dimer.

How long is the actual miRNA in the library?

  • Inserts can be up to ~50 bases in size, as this is usually the upper size cutoff due to the bead-based size selection. Sequencing at 1 x 72 supports sequencing into the UMI region.

Will the Illumina miRNA library prep kit and associated indexes be added to the BaseSpace Run Planner?

  • Yes.

What are the override cycles used for UMI? (seen in BaseSpace Run Planner)

  • Read1: Y72

  • Index1: I10

  • Index2: I10

Will there be demo datasets for runs in BaseSpace?

  • Yes, to be published by product launch. All Illumina demo datasets can be found in BaseSpace Demo Data.

For any feedback or questions regarding this article (Illumina Knowledge Article #9895), contact Illumina Technical Support [email protected].

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