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Old 05-13-2018, 05:12 PM   #32
idedios
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Location: Irvine, CA

Join Date: Mar 2014
Posts: 18
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Quote:
Originally Posted by korostin View Post
We aren't looking for easy ways)) Price per 1 library more 100$ it's too expensive
Yeah I've been looking at alternatives too but it's hard to beat the sequence quality and workflow simplicity.

I'll try out a DIY ampliseq approach using NEB enzymes with some modifications:
1. OneTaq Hot Start Master Mix with GC buffer should be sufficient for the multiplex PCR reaction with uracil-containing ampliseq primers. I would scale the reaction down to 20 ul and run a 4/8/16 minute anneal extend. NEB recommends a 68C extension however such a long annealing should provide enough extension activity. Also annoying that Thermo calls their PCR enzyme "AmpliSeq Hifi" when it probably isn't even a high fidelity enzyme.
2. There was a post suggesting using USER and Exonuclease I for the FuPa digest however it was never tested and I'm not sure what the difference is between USER and UDG. I would simply add 10 units of UDG and Endonuclease IV directly to each PCR reaction and incubate at 37C for 1 hour. Adding 3' dA tailing would be convenient for Illumina adapter ligation
3. Blunt/TA Ligase master mix, scaled up to ~ 50 ul though it might be possible to find a 5x mix to keep the ratios as similar to Thermo's as possible. This would work with Ion Xpress adapters or off-the-shelf Illumina barcoded adapters if dA-tailing was performed.
4. Ampure wash, same as Thermo's recommendation but scaled up.
5. Library amp with Q5 Hot start HiFi 2x master mix and P1/X primers or P5/P7 primers.
5. Final size selection and ampure wash as per Thermo's guidelines.

The total cost of the NEB enzymes came out to around $8 per sample. I would order custom P1/X primers from IDT as well as off-the-shelf P5/P7 primers off IDT. Most expensive components would be the AmpliSeq primers followed by the sequencing adapters. The total cost per sample would still be far less than the $100/sample Thermo and Illumina are charging for without the AmpliSeq primers or barcoded sequencing adapters.

Let me know if I'm missing anything.

Ivan
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