Workflow and Sequencing recommendations FAQ for the TruPath Genome assay

Note: Trupath Genome is compatible with the NovaSeq X instruments only, with a minimum control software version of 1.4.

What is the mechanism by which TruPath Genome libraries are made?

Library prep uses flow cell-bound transposomes which capture and tagment long molecules of dsDNA as they are flowed onto the flow cell surface. Clustering and SBS are as standard, resulting in high-quality short-read sequencing.

What changes to the sequencing workflow are required for TruPath?

Flow cell prep mix (FP1) should be added to CP1 position and Flow Cell Transposome (FT2) should be added to CP2 position. See the Product Documentation for full details.

Can FP1, FT2, and FTB reagents be refrozen after thawing?

If running on the same day, keep these reagents at 4°C for up to 4 hours. Otherwise, remove these reagents from the cartridge, and store in capped tubes at -20°C until subsequent use. Up to 5 freeze-thaws are supported.

How many samples are supported per sequencing run?

2 samples per C2 flow cell and 8 samples per C8 flow cell (1 sample per lane for both flow cells). Therefore, double per run with 2 flow cells on a NovaSeq X Plus; 16 samples per C8 run and 4 samples per C2 run.

Can TruPath Genome runs utilize NovaSeq X v1.4 staggered start?

TruPath Genome runs can be staggered with another TruPath or any other regular flowcell on the NovaSeq X.

What is the recommended read length?

The recommended read length is 2x151. Run setup in BaseSpace Run Planning requires 2x151 run set up with no indexes (use "0" for index1 and index2).

Are indexes sequenced**?**

TruPath does not require indexing, as only one sample per flow cell lane is supported.

Is PhiX required? What happens if it is added?

No. PhiX DNA or PhiX library (eg, Illumina PhiX control v3, cat # FC-101-3001) must not be added to the human gDNA samples.

Is a custom recipe required?

C2 and C8 flow cells will have a RFID to load the correct recipe, this will require the V1.4 software installed on the NovaSeq X to work.

A custom recipe cannot be loaded when running a C2 or C8 flow cell.

Does the above require selecting custom sequencing primers? Ie, should the check boxes for custom primer use be selected, during run setup?

No, do not select the check boxes (they must be kept blank) for Custom Primers during run setup. The flow cell RFID will load the correct sequencing recipe for TruPath Genome libraries, which instructs the instrument to use the contents of the CP1 and CP2 wells at the appropriate times. The instrument software will throw an error if custom primers, custom recipes, or indexes are selected for runs using TruPath flow cells.

What is the total sequencing time?

Both C2 and C8 TruPath Genome sequencing runs will complete within 29 hours.

Is there anything specific required for the BCL Convert settings for TruPath sequencing ›runs?

• Read lengths must be: 151, 0, 0, 151

• Library prep kit: choose one:

  • Illumina TruPath Genome Prep (C2, 2 samples)

  • Illumina TruPath Genome Prep (C8, 8 samples)

• Index Adapter kit: Not Applicable

• For physical run setup, customers can only sequence 1 DNA sample per lane.

What is the phenotype if there is a swap of the reagents within the CP1 and CP2 positions?

Sequencing run will fail. If the swap is detected prior to loading the cartridge onto the sequencer, reagents can be removed and placed back into their respective tubes. CP1 and CP2 wells should then be washed three times with 4 ml water, before adding the reagents back to the correct positions.

What are the sequencing expectations for TruPath Genomes runs?

The expectations for TruPath Genome sequencing runs are as follows:

Flow cell	%Q30	Yield/Lane	Yield/Total
C8	85%	241 Gb	1928 Gb
C2	85%	241 Gb	482 Gb

For any feedback or questions regarding this article (Illumina Knowledge Article #10168), contact Illumina Technical Support [email protected].