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Old 05-07-2019, 05:47 PM   #1
SDPA_Pet
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Location: US

Join Date: Apr 2013
Posts: 222
Default Basic questions about Illumina Amplicon sequecing

Hi, I've just moved to a new lab. We normally do barcoded bacterial 16S rRNA and fungal ITS sequencing. However, this new lab's protocol and primers design are quite different from my previous lab's. I'm confused about this.

1> I have an excel spread sheet about list of each barcode + primers? There is one column called "Pad". I have 96 barcodes, but the "pad" sequence are all same like this "TATGGTAATT". What does Pad stand for? Primer Pad? What is the function for this? I don't remember my previous lab uses this pad? Do I have to add this in my barcode primers? what is the benefit for adding a pad sequence?

2> There is also a column called "linker". Again, all linker are same as "GT"? Again, what is the function for this? what is the benefit for adding a linker sequence?

3> The new lab's protocol is different from my old lab. Our old lab uses two-step PCR (see details here: https://www.researchgate.net/profile...g+Approach.pdf)

However, the new lab uses one step PCR added everything P5/P7 adapters + primers+ pad+ linker+ barcode.

what confused is the next step, when we submit to sequencing center. My new PI also ordered so-called sequencing primers and told me that sequencing center needs these. I check the these sequencing primers. Basically, they are my Forward primer+P5 adapter and reverse primer+P7 adapter?

Can anyone tell me what these for in sequencing center? I suppose the sequencing center will do a bridge amplification before start real sequencing!!!

I maybe had a bad memory. I don't think we had this in my old lab. I was told by my old PI that sequencing center will use universal primer to do bridge amplification. I don't know why the new lab will give them the specific primers. Is this optional, but specific primers are always better?
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