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Old 01-18-2016, 09:58 AM   #5
kmcarr
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Location: USA, Midwest

Join Date: May 2008
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Quote:
Originally Posted by wisnu.wicaksono View Post
Hi guys,

Thank you so much for the answer. I just want to confirm whether we will have the same conclusion or no. How about if we do amplicon sequencing that we know the lenght of amplicon for example 250 bp. Is there any possibilities that the sequencing primer can read the whole sequence include the barcode?thank you and have a nice day
But can you really know the ALL of your amplicons are 250bp? For example the 16S V4 region (515-806) is nominally 253bp (after trimming primers) but there is species dependent length variation.

If your library is designed to use standard Illumina sequencing and index primers then just set up your run to use them. They are already included in the sequencing reagents. If your are designing a library product which will require custom sequencing primers then just order the custom index primer; primers are dirt cheap compared to all the other costs in your experiment. If you will already be ordering custom read 1 and read 2 primers then you really only need to add index primer 1 which most often is just the reverse complement of the read 2 primer. (This is based on standard library designs, I can't say for certain that this will be true in your case.) For paired-end, dual indexed sequencing, index 2 is primed using the flowcell bound oligo so there is no need to order index 2 primer. (Again, based on standard designs, YMMV.)
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