Sequencing FAQ for the Illumina 5 Base WGS and Enrichment kits

Which instruments are supported for sequencing of this library type?

• NovaSeq 6000: S4 standard or Xp loading

• NovaSeq X: 1.5B, 10B, 25B

• NextSeq 1000/2000: XLEAP P3 onboard denature and dilute

What is the recommended sequencing read lengths and override cycles?

• 2x 151 with dual, 10 bp indexes

• Override cycles are U7N1Y143;I10;I10;U7N1Y143

• IMPORTANT: omitting the override cycles will lead to higher-than-normal false positive rate (typically around 3.5-4%). This is because indexes and UMI anchors are not methylated.

What are the recommended loading concentrations?

The following loading concentrations apply to WGS libraries as well as Enriched libraries. These are general starting guidelines and loading concentrations may need to be adjusted/optimized for optimal performance.

• NovaSeq 6000, S4 standard = 0.5 nM (starting concentration), 100 pM (final concentration)

• NovaSeq 6000, S4 Xp = 0.25 nM (starting concentration), 50 pM (final concentration)

• NovaSeq X, 1.5B = 130 pM

• NovaSeq X, 10B = 130 pM

• NovaSeq X, 25B = 150 pM

• NextSeq 1000/2000, XLEAP P3, onboard = 750 pM. Note: NextSeq 1000/2000 output is insufficient for WGS libraries and is intended for Enrichment libraries

Can samples from gDNA and cfDNA be pooled together for sequencing even though the fragment sizes differ?

• This is not recommended to ensure optimal sequencer performance.

Is it recommended to add PhiX to the sequencing run? If yes, how much?

• Yes, 1% for all sequencing runs.

Can a 2% PhiX spike-in be used instead of the recommended 1%?

• Yes, if preferred. More reads will go to PhiX at 2% compared to 1%.

What sequencing depth/number of reads per sample is recommended? What is the recommended samples per run?

For WGS (see datasheet):

• NovaSeq 6000, S4 (standard or XP) = 18 libraries per flow cell

• NovaSeq X, 1.5B = 3 libraries max

• NovaSeq X, 10B = 18 libraries max

• NovaSeq X, 25B = 48 libraries max, either single pool across all lanes, or 6 libraries per lane

For Enrichment

• For gDNA enrichment: target 25 reads (clusters) per bp of target panel

• For cfDNA enrichment: target 200 reads (clusters) per bp of target panel

What sequencing coverage depth does Illumina recommend for various applications?

• Illumina recommends 35X coverage for optimal performance of methylation calling + variant calling. If interested in methylation calling only, can consider 30X or even lower.

• For other applications, see the below for suggested starting coverage depth recommendations, some optimization may be required (information from the datasheet):

  

If sequencing a pool of samples, is it better to make one pool and split it across all lanes, or sequence specific (and different) samples per lane?

• Both options are valid if aiming for the same number of reads per sample.

• However, if performing unequal pooling (some samples loaded at higher volume/concentrations than others), then the recommendation is to split up pooling events into smaller pools split across the flow cell (not recommended to do a single pool per flow cell in this scenario). If performing unequal pooling and a single sample is pooled significantly higher than the others, resulting in the majority of the clusters in all lanes, it can cause data processing issues downstream.

Is it better to run eight libraries per flow cell and run three flow cells, or make one pool of 24 libraries, and sequence it three times?

• It is better to make a pool of the 24 libraries and sequence it three times. This allows for rebalancing after the first run, though it is important ensure there is sufficient library in for re-pool, if needed.

Do the 5-Base runs have a different base profile compared to standard whole genome or enrichment runs?

• Generally, no. In human genomes, most methylation is observed in the CpG context only. There are ~28 million cytosines in the CpG context in the human genome (~3.3 billion bases). Thus only ~1% of the genome is available to be converted from 5mC>T. This will not be noticeable when reviewing run performance metrics.

For any feedback or questions regarding this article (Illumina Knowledge Article #9949), contact Illumina Technical Support [email protected].