lots of short read between 16s rDNA Region V1-V3 ?

[stare024]

I did 15 runs of 16s rDNA Amplicon in passed few days，
and 2.5 run within 16s rDNA region V1-V3，which the amplicon about 500bp long，
but there lots of short reads about 250-300bp，60-100bp，（sequence from A-end）
I selected ten short reads ，and blast to NCBI show that all match the 16s rDNA except the 3‘-end，about 10-15 bases，which seems the sequence of the primer B .

I wonder that does the primer B will generate non-specific amplification between 16s rDNA region v1-v3 ？

[SeQnewby]

GS Junior-multiple read lengths after size selection

Hi, I also have problems with multiple read lengths.

I'm testing out new 16S pyro primers. It's a normal unilateral 454 run and the products were size selected on a 2% gel prior to emPCR. The size is 450bp with forward and reverse primers being 55bp in size. The control beads showed a perfect run with the correct size however my run has two additional read length peaks at 250 and 350bp. I have no idea why but I'm suspecting the primers.

Has anyone run into this same problem?

Please help*

[elizondas]

Hi,
What kind of primers did you use to amplify V1-V3? Tha same as HMP?

[SeQnewby]

Hi elizondas

I don't know about stare but I used primers amplifying the V3-V5 region due to the large overestimation V1-3 gives you in species diversity. SILVA published a paper regarding the best current primers for various applications and these I modified for pyrosequencing.

My first thought was that it's the reverse primer acting up during emPCR, but I have yet to check if its the case. Someone at one of the other forums said this could happen if your reverse primer tag has a high homology to your target region. But offcourse you can't change your tag sequences-which complicates things....

[elizondas]

Thanks. I would like to sequence 16s from more than 400 sample of nasopharyngeal samples by pyrosequencing, and I have tought about V1-V3 primers based on some papers.

We want to use 27F-534R primers to sequencing V1-V3 region. I had thought to add them adaptor A and B, and then a Broad Institute bardcode in reverse primers.
Do you have have any experience in this? direct experience in issues as sample preparation, quality control etc