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Old 06-21-2013, 12:15 AM   #1
stare024
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Location: suzhou city china

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

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 ?
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Old 09-09-2013, 05:47 AM   #2
SeQnewby
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Location: South Africa

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Default 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*
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Old 11-25-2013, 02:37 AM   #3
elizondas
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Hi,
What kind of primers did you use to amplify V1-V3? Tha same as HMP?
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Old 11-25-2013, 02:58 AM   #4
SeQnewby
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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....
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Old 11-25-2013, 03:08 AM   #5
elizondas
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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
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