Sequencing Considerations

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

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

Library Type	Loading Concentration (pM) for Standard SBS Kits
AmpliSeq for Illumina Library PLUS	750
Illumina DNA Prep	750
Illumina DNA Prep with Enrichment	1000
Illumina DNA Prep with Exome 2.5 Plus Enrichment	1000
Illumina Stranded Total RNA with Ribo-Zero Plus	750
Illumina Stranded mRNA Prep	750
Illumina DNA PCR-Free	1000
TruSeq DNA Nano 350	1200
TruSeq DNA Nano 550	1500
TruSeq Stranded mRNA	1000
100% PhiX	650

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.

Number of Cycles in a Read

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 for 100-cycle, 200-cycle, and 600-cycle kits and 27 cycles for P3 300-cycle kits. 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:

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For a read length of 35 (single-read), enter 36 in the Read 1 field.

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For a read length of 150 per read (paired-end), enter 151 in the Read 1 field and 151 in the Read 2 field.

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For a read length of 300 per read (paired-end), enter 301 in the Read 1 field and 301 in the Read 2 field.