Standard vs Low Input Workflow selection FAQ for Illumina DNA PCR Free prep

What happens if the input mass is below the saturation; will the library prep still work with similar quality?

• For 100 ng to 299 ng of DNA input, the library preparation method is the same as when saturation is achieved. The only difference is the need to quantify the final libraries before pooling by qPCR to verify sequencer loading, as library normalization with input below 300 ng is not achieved.

• For 25-99 ng DNA input, use the the low input library protocol; library yield must be assessed by qPCR before pooling, as library yields are not normalized. Due to changes in size selection step of low input protocol, certain sequencing metrics (such as Q30 and insert size) may be slightly lower comparable to standard protocol prepared libraries.

• See the “DNA Input Recommendations” section of the Reference Guide for more information.

Why, in the standard input workflow, are the BLT-PF and TB1 added separately, while, for the low input workflow, a master mix is made? Pre incubation of BLT-PF with TB1 activates the BLT-PF, enhancing library quality for low inputs; however, this can reduce library yield for inputs >99 ng. Therefore, Illumina recommends different orders of addition for the different protocols, as indicated in the Reference Guide.

If the standard workflow is used for low input DNA, can the libraries be sequenced? Using the standard workflow with low input results in lower library yields, though libraries can still be sequenced if sufficient library yields was generated.

If the low input workflow for high input DNA, can the libraries be sequenced? Library quality will be compromised, contact Illumina Technical Support for more information.

Can the low input workflow be used in combination with the saliva, DBS, or whole blood workflow?

This is not recommended for these sample types, even for lower yields from these samples types.

For any feedback or questions regarding this article (Illumina Knowledge Article #3460), contact Illumina Technical Support [e-mail].