Hi Everyone,
I was just thinking about things that might affect the results of 16S amplification and sequencing in terms of lab-based biases. Do you take any of these into account when designing and executing experiments? Have you ever measured their effects?
1. Real base balance in oligo synthesis where an ambiguity is specified: Bases don't have equal coupling efficiencies, so 'N' in the oligo order doesn't necessarily equate to 25%A, G, C, T at that position across the oligos in the pool).
2. Primer synthesis termination and deletion products: As little as 30% of synthesised oligo is full-length and is the expected sequence. The rest is early termination products and deletion products.
3. Amplification efficiency/exponential amplification/reaction kinetics - It seems that there's a much higher likelihood of chimera formation, template amplification suppression and introduction of other biases when the reaction is 'inefficient' for whatever reason (such as 'tired' PCRs where reagents are running low in the reaction due to excessive cycling).
4. Non-specific amplification.
5. Polymerase choice and enzyme error profiles.
... anything else you consider to be a problem? What else do you evaluate, measure and/or control?
Cheers,
Scott.
I was just thinking about things that might affect the results of 16S amplification and sequencing in terms of lab-based biases. Do you take any of these into account when designing and executing experiments? Have you ever measured their effects?
1. Real base balance in oligo synthesis where an ambiguity is specified: Bases don't have equal coupling efficiencies, so 'N' in the oligo order doesn't necessarily equate to 25%A, G, C, T at that position across the oligos in the pool).
2. Primer synthesis termination and deletion products: As little as 30% of synthesised oligo is full-length and is the expected sequence. The rest is early termination products and deletion products.
3. Amplification efficiency/exponential amplification/reaction kinetics - It seems that there's a much higher likelihood of chimera formation, template amplification suppression and introduction of other biases when the reaction is 'inefficient' for whatever reason (such as 'tired' PCRs where reagents are running low in the reaction due to excessive cycling).
4. Non-specific amplification.
5. Polymerase choice and enzyme error profiles.
... anything else you consider to be a problem? What else do you evaluate, measure and/or control?
Cheers,
Scott.
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