Sequencing Considerations

This section is only applicable to protocols utilizing XLEAP-SBS chemistry. If your protocol utilizes standard SBS chemistry, refer to Standard SBS Sequencing Protocol.

Before starting the protocol, review the following information to prepare for diluting libraries and setting up the run. Achieving the optimum loading concentration is critical for successful sequencing and analysis. Entering the correct number of cycles in a read helps ensure optimum data output.

For information on planning a run in BaseSpace Sequence Hub, refer to Illumina Cloud Run Planning.

Loading volume is 20 µl. Loading concentration varies by library type:

Library Type

Flow Cell

Loading Concentration (pM) for XLEAP-SBS Kits

Illumina DNA Prep with Enrichment

All

1000

Illumina Stranded Total RNA with Ribo-Zero Plus

All

750

TruSeq DNA Nano 550

P1/P2 (600-cycle)

1500

 

 

100% PhiX

P1/P2 (2 x 151 or less cycles)

650

P1/P2 (2 x 300 cycles)

450

P3/P4

450

For other library types, 650 pM is the recommended starting loading concentration. Optimize this concentration over subsequent runs to identify a loading concentration that consistently yields data that meets specifications.

To optimize loading concentration, use the % Loading Concentration metric in the PrimaryAnalysisMetrics.csv output file available after run completion. If % Loading Concentration is < 95%, increase the loading concentration in 100 pM increments over subsequent runs.

For each read, entering a minimum of 26 cycles and a maximum of 301 cycles helps ensure data quality. The exact number of cycles depends on your experiment. NextSeq 1000/2000 Control Software requires at least 1 cycle for Read 1, but displays a warning when the number of cycles in Read 1 is less than 26.

The total number of cycles for Read 1, Index 1, Index 2, and Read 2 cannot be greater than the number of cycles supported by the kit plus 38 cycles. NextSeq 1000/2000 Control Software displays a warning when Index 1 and Index 2 are fewer than 6 cycles. The warning is not shown if Index 1 or Index 2 is 0 cycles.

The minimum and maximum cycle numbers include an extra cycle. Always add one cycle to the desired read length to correct the effects of phasing and prephasing. Read length is the number of sequencing cycles in Read 1 and Read 2, which excludes extra cycles and index cycles. For more information, refer to Phasing Correction in Real-Time Analysis Workflow.

Example run setup:

• For a read length of 35 (single-read), enter 36 in the Read 1 field.
• For a read length of 150 per read (paired-end), enter 151 in the Read 1 field and 151 in the Read 2 field.
• For a read length of 300 per read (paired-end), enter 301 in the Read 1 field and 301 in the Read 2 field.