Saliva processing for Illumina DNA PCR Free library preparation workflow questions

Is the saliva extraction kit used here the same as used for Illumina DNA Prep, (M) Tagmentation (formerly Nextera DNA Flex)? The same tube is used to collect saliva. (Saliva in is collected in Oragene tubes.)

Following saliva collection, how long are samples stable? Samples are stable at room temperature for up to 5 years after the 50°C incubation step.

Can saliva samples be frozen? Oragene/saliva samples can be stored at -20°C or -80°C. Samples can undergo up to three freeze-thaw cycles with no evidence of DNA degradation.

Does the 50°C incubation of the saliva sample need to be performed immediately prior to starting the lysis protocol? How long does the 50°C incubation need to be?

• No, the incubation can be performed at any point prior to beginning the prep. Following the 50°C incubation step the sample is stable at room temperature for up to 5 years.

• Incubated samples for at least 1 hour, up to overnight.

What happens if more or less than 50 µl of saliva is added to the lysis reaction?

This is not recommended, as it may affect the efficiency of the lysis reaction which will ultimately impact the amount of DNA obtained and therefore the final library yield. Because beads may not be saturated with less than 50 µl input, libraries must be quantified individually.

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