Input Recommendations

The following section provides input recommendations.

Assess RNA Sample Purity

IMAP-Flu supports RNA inputs from various sample types. To improve library preparation success, use the highest quality nucleic acid input. It is good practice to assess sample quality before beginning targeted library prep.

UV absorbance is a common method used for assessing the purity of a sample. The ratio of absorbance at 260 nm to absorbance at 280 nm provides an indication of sample purity. This protocol is optimized for RNA with 260/280 absorbance ratio values of 2.0–2.2, which indicates a high purity sample.

For a secondary indication of sample purity, use the ratio of absorbance at 260 nm to absorbance at 230 nm. Target a 260/230 ratio of 2.0–2.2. Values outside this range indicate the presence of contaminants. Using nucleic acids that do not meet these recommendations may result in assay failure. If you experience assay failure, Illumina recommends cleaning up the samples and repeating library preparation.

Purity Check

RNA

A260/280

2.0–2.2

A260/230

2.0–2.2

Given the highly variable target abundance in sample types used with this technology, Illumina recommends using the CDC qRT-PCR protocol¹ or the Ward protocol² for measuring influenza A and influenza B viral loads, and samples with Ct values < 30 for high sequencing depth and complete whole genome coverage.

Many commercially available RNA extraction and purification kits are available. Choose a kit or method that produces the highest quality nucleic acids possible for your specific targets and from your specific sample types. Illumina tested the following kits. Other kits or methods may be preferred for your samples and targets.

• QIAamp Viral RNA Mini Kit, QIAGEN, part# 52906 and 52904.

1. Bo S, Kai-Hui W, Shannon E, et al. Design and Performance of the CDC Real-Time Reverse Transcriptase PCR Swine Flu Panel for Detection of 2009 A (H1N1) Pandemic Influenza Virus. Journal of Clinical Microbiology. Published July 2011. Accessed September 15, 2023. https://journals.asm.org/doi/10.1128/jcm.02636-10.

2. Ward CL, Dempsey MH, Ring CJA, et al. Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement. Journal of Clinical Virology. Published March 2004. Accessed September 15, 2023. https://www.sciencedirect.com/science/article/pii/S1386653203001227.