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Old 06-27-2013, 03:32 PM   #1
Kim_2013
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Default Barcoded primers for PacBio sequencing

Hi

I am going to use PacBio sequencer to sequence some community samples

I plan to take a variable region from 16S rRNA gene. I decided the primers but i am struggling with deciding the barcode.

I have many samples, and plan to use asymmetric barcodes for the insert of about 500bp. PacBio guideline recommends to use the recommended barcodes (they provided a recommendation list of about 48 pairs of 16 bp barcodes) but i really don't have idea how can i decide which barcode should be chose?

I checked the secondary structure of barcoded primers using http://www.idtdna.com/pages/scitools but don't know how can i judge the parameters? (self dimer, hair pin...)

Anyone can explain and teach me?. thank you so much!
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Old 06-27-2013, 10:58 PM   #2
flxlex
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First, I would use a longer part of 16S then 500 bp, you may even try the whole 1.5 bkp with today's read lengths! See http://flxlexblog.wordpress.com/2013...us-sequencing/ :-)
Second, unless you need more than the set of barcodes that PacBio suggests, I would trust their list, surely they have tested for self-dimers, hairpins etc.
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Old 06-28-2013, 10:33 AM   #3
Kim_2013
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Thanks flxlex for your response. A week ago, I already read the link you sent.

I decide to use only 500bp since i am going to do community analysis. I think the higher number of 500 short sequence is better than the small sequence of full length.
I do trust the barcodes suggested by PacBio, I only concern about the barcoded primers (primers with bacode attached). How can i decide based on the test for self dimer, hairpin...?

Or Can i pick any recommended barcode for my primers?
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Old 08-07-2013, 04:08 AM   #4
bstamps
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If you plan on only amplifying 500bp, I might ask why you aren't using Illumina and overlapping each read 50bp and then stitching the reads to get a final artificial read near 500bp (Or waiting a few months and getting 500bp when they move to 2x300). Not to knock pacbio, we use it for genome finishing, but amplicon sequencing for community analysis was made for the Illumina platform.

If you did plan on using the PacBio RS I would agree with flxlex and go with full length 16S. You would get considerably higher resolution of your community and take advantage of what PacBio does best (Long reads).
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