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Old 05-07-2019, 05:47 PM   #1
SDPA_Pet
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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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Old 05-09-2019, 04:35 AM   #2
kmcarr
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It sounds like your new lab is following the protocol developed by Patrick Schloss' lab (or something very similar) for construction dual indexed Illumina amplicon libraries. You can review the full details of the protocol here. Our core follows this protocol for 16S-V4 amplicon library prep and it's very reliable.

Regarding the Pad and Linker, these are described in Appendix C (Primer Design) of that document:

Quote:
The pad is a 10-nt sequence to boost the sequencing primer melting temperatures. The link is a 2-nt sequence that is anti-complementary to the known sequences.
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Old 05-09-2019, 01:06 PM   #3
SDPA_Pet
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Thanks. Do you have any idea about the so-called sequencing primers?
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Old 05-09-2019, 01:36 PM   #4
kmcarr
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Quote:
Originally Posted by SDPA_Pet View Post
Thanks. Do you have any idea about the so-called sequencing primers?
The custom sequencing and index primers are complementary to the target specific portions of the original PCR primers, plus the pad and linker. For example, if the amplicon you are sequencing is 16S-V4 generated with the usual 515F forward primer then the custom R1 sequencing primer would be:

<pad><link><515F>

or

TATGGTAATT GT GTGCCAGCMGCCGCGGTAA

These are all described in the WetLab SOP, Appendix C referred to in my post above. (Look for "Generic read 1 primer design", etc.)

The reason custom sequencing primers complementary to the 3' ends of your PCR primers are used is so that you aren't wasting sequencing data reading through your PCR primers which you would then need to trim off the 5' ends of your reads. This method was also used in the protocol developed in the Knight lab for their 16S MiSeq libraries. Figure 1 in the reference below gives a good presentation of the positions of these sequencing and index primers and their relationship to the ends of the PCR primers.

Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences 108 Suppl 1, 45164522 (2011).
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Old 05-09-2019, 02:22 PM   #5
SDPA_Pet
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Thanks. I just want to make sure if the sequencing primers for bridge amplification step for Illumina sequencing? Am I right? I just want to know which step the sequencing center would use these sequencing center.

When I check the youtube video about illumina sequencing, I don't think illumina requires a sequencing primers to sequencing.

Last edited by SDPA_Pet; 05-09-2019 at 02:27 PM.
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Old 05-10-2019, 04:00 AM   #6
kmcarr
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Quote:
Originally Posted by SDPA_Pet View Post
Thanks. I just want to make sure if the sequencing primers for bridge amplification step for Illumina sequencing? Am I right? I just want to know which step the sequencing center would use these sequencing center.

When I check the youtube video about illumina sequencing, I don't think illumina requires a sequencing primers to sequencing.
Yes, Illumina sequencers absolutely require sequencing and index read* primers. Bridge amplification and sequence generation are separate things. Every Illumina reagent kit/cartridge has tubes/wells of dedicated read 1 (R1), read 2 (R2) and index 1 (I1) primer mixes. Your custom primers will be mixed into those. Illumina provides specific guidelines for the concentration of custom primers to mix in. Make sure that your sequencing provider is familiar with adding custom primers to a reagent kit/cartridge.

*The exception to separate primers is for index read 2 (I2) in dual index situations. That read is primed using the oligos grafted to the flow cell surface. That's why the dual index 16S-V4 protocol includes custom primers for R1, R2 and I1 but not I2
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