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SDPA_Pet 02-20-2019 03:09 PM

MiSeq amplicon sequencing Primers?
 
Hi guys,

I sent some soil samples to a commercial sequencing center. I do fungal ITS sequencing (300bpX 2 paired-end) Miseq.

As you know, I use forward and reversed primers when I did PCR. I suppose Paired-end means sequencing twice, right? I suppose they should use forward primers to sequence once and reverse primer to sequence from another time.

However, I received two mapping files from sequencing center. Both mapping file show they only used forward primer? I am wondering if this is normal?

I know in our forward primer always better and accurate. Back to old days (Sanger sequencing) we only use forward primers to run sequencing reactions.

If you guys are familiar with paired-end amplicon sequencing, I am wondering if it is normal to use only one primer to sequence both (paired).

Thanks,

nucacidhunter 02-20-2019 05:27 PM

I assume you have done PCR wit ITS region specific primers with no Illumina overhang or full adapter sequences. In this case they must have prepared library by adapter ligation which will result in non-direction library so R1 and R2 can start for some fragments from forward direction and for others from reverse direction.

You should check some reads from R1 and R2 sequence folders and you should see in both sequences starting with forward and reverse primers, unless they have been sequenced with custom primers or have been trimmed to remove PCR primers.

SDPA_Pet 02-20-2019 06:09 PM

Quote:

Originally Posted by nucacidhunter (Post 224107)
I assume you have done PCR wit ITS region specific primers with no Illumina overhang or full adapter sequences. In this case they must have prepared library by adapter ligation which will result in non-direction library so R1 and R2 can start for some fragments from forward direction and for others from reverse direction.

You should check some reads from R1 and R2 sequence folders and you should see in both sequences starting with forward and reverse primers, unless they have been sequenced with custom primers or have been trimmed to remove PCR primers.

I don't quite understand, but I didn't do PCR. I gave them gDNA. They did the PCR and sequencing for me.

They didn't give me raw fastq files such as R1 and R2. They only give me a combined fasta file. I only find adapter sequence and forward primer sequences in the fast file. I don't they if they removed reversed primers or they only did sequencing using forward primer.

nucacidhunter 02-20-2019 06:31 PM

Paired end sequencing means that library fragments (in this case ITS amplicons) are sequenced from both ends so R1 and R2 of the fragment are in opposite direction. If sequencing cycles are longer than library fragment then R1 nad R2 can be merged (if there is enough overlap). You can ask the sequencing centre to provide at least the workflow and that should answer your question.

SDPA_Pet 02-20-2019 06:37 PM

Quote:

Originally Posted by nucacidhunter (Post 224109)
Paired end sequencing means that library fragments (in this case ITS amplicons) are sequenced from both ends so R1 and R2 of the fragment are in opposite direction. If sequencing cycles are longer than library fragment then R1 nad R2 can be merged (if there is enough overlap). You can ask the sequencing centre to provide at least the workflow and that should answer your question.

ôR1 and R2 of the fragment are in oppossite direction". -- I know this. However, my question is when they sequence R1 and R2, do they have to use two primers? For example, R1 uses forward primer and R2 uses reverse primer.

Or they even don't have to use the PCR primer? They use universal primers?

nucacidhunter 02-20-2019 08:54 PM

R1 and R2 are sequenced with different primers. Sequencing primers could be one of Illumina sequencing primers or custom primers based on amplicon sequence. The primer choice is based on library prep workflow and design.


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