Oligo quality issue

[HMorrison]

Had 32 primers made that started with "NNNN" at 5' end. I have a full lane of Illumina data reading through these bases and into an inline bar code. I almost always recover the bar code perfectly. I expected to see each base at ~25% in these cycles; instead I get 16%, 17%, 30% and 37% for A, C, G, and T, respectively. That's some pretty sloppy work by the company. I haven't made oligos myself in years, but isn't is just a matter of mixing the four bases in a 5th bottle "N" that goes on the synthesizer?

Interesting what you learn now that sequencing through oligos as single molecules common. Had a synthesis by a different company (one that is usually highly recommended) where the 3' base was incorrect. They are synthesized from 3' end.

[pmiguel]

I think some companies offer to "balance" the nucleotides in an "N" base for an additional fee. So I don't think it is just a matter of spiking in the right molar concentration of the reagents.

That said, seems like many presume that oligo synthesis has far lower (or zero) error rates than it actually has.

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Phillip

[HMorrison]

I think some companies offer to "balance" the nucleotides in an "N" base for an additional fee. So I don't think it is just a matter of spiking in the right molar concentration of the reagents.

That said, seems like many presume that oligo synthesis has far lower (or zero) error rates than it actually has.

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Phillip

This company says "Mixed Bases* Degenerate bases. Equal amounts of the designated bases are delivered by the synthesizer.
A+C+G+T N"

If a company charged extra for "balancing", I would shop elsewhere. I don't expect perfection, but this is way off the mark. On average, I want the population to reflect what I've ordered. I don't think that is unreasonable or naive.

[pmiguel]

This company says "Mixed Bases* Degenerate bases. Equal amounts of the designated bases are delivered by the synthesizer.
A+C+G+T N"

If a company charged extra for "balancing", I would shop elsewhere. I don't expect perfection, but this is way off the mark. On average, I want the population to reflect what I've ordered. I don't think that is unreasonable or naive.

Many things are easier said than done. If I really needed a balanced N oligo, I would probably take the fact that a company is charging more for it as a sign that they realize there is an issue.

But, you may be right. Maybe you just got a "Monday" batch of oligos.

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Phillip

[HMorrison]

Many things are easier said than done. If I really needed a balanced N oligo, I would probably take the fact that a company is charging more for it as a sign that they realize there is an issue.

But, you may be right. Maybe you just got a "Monday" batch of oligos.

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Phillip

Not sure what company you use. I find that option on the IDT site, although they still offer the vanilla mixed base options using IUPAC codes. I then had a chat with one of the IDT staff. The costs for hand mixed reagents are high. Machine mixing is known to be biased: "Actual reactivity favors T>G>C>A (30%:26%:24%:20%)" and differential flow rates can skew it further. It has been a good day for learning new ways to bias your PCR and sequencing results! Guess the days of the 5th bottle are over (makes sense, now that I think about it more carefullly).

[pmiguel]

Not sure what company you use. I find that option on the IDT site, although they still offer the vanilla mixed base options using IUPAC codes. I then had a chat with one of the IDT staff. The costs for hand mixed reagents are high. Machine mixing is known to be biased: "Actual reactivity favors T>G>C>A (30%:26%:24%:20%)" and differential flow rates can skew it further.

Maddening, isn't it? And your results do suggest higher skews than those.

It was one of our customers attempting to use Sanger sequencing chromats to assay the effects of a molecular evolution study who mentioned the availability of higher cost "balanced" N oligos.

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Phillip