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Old 09-05-2013, 11:20 AM   #1
ChristmasSunflower
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Default 16S rRNA amplicon sequencing kit

I wonder if anyone knows any commercial kit available currently for 16S rRNA amplicon sequencing? I know we can design customized primer for this. But this is a pilot experiment to generate preliminary data for grant proposal so I like to keep this simpler. Ideally I'm looking for the kit to amply V1-V2 region of 16S rRNA gene. The sequencing will be done by MiSeq either 250X2 or newly 300X2 kit.

Any information will be much appreciated.

Thanks
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Old 09-05-2013, 11:50 AM   #2
microgirl123
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This (http://seqanswers.com/forums/showthread.php?t=32475) thread has a method that is relatively inexpensive and easy and will let you use any amplicon. BioO sells a 16s kit, but it's the V4 region.
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Old 09-05-2013, 12:31 PM   #3
ChristmasSunflower
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Thanks microgirl123. I notice BIOO offers V4 kit. I try to avoid to design the sets of primers for this pilot experiment, wonder if there are commercial kits available.
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Old 09-06-2013, 07:30 AM   #4
microgirl123
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I think you'll have trouble finding a kit for V1-V2 for the MiSeq since the metagenomics workflow on the MiSeq is designed for V4. This (http://www.humgenomics.com/content/p...64-4-3-147.pdf) paper has published V1-V2 primers that you could use in combination with the method I linked to above (as long as the amplicon is short enough for the MiSeq to fully sequence it). I'm sure there are other published primer sets for that region also.

You just need to order one set of the V1-V2 primers with flanking partial adapter sequences and do one round of PCR. Then you take that product and do another 5 cycles of PCR with Nextera adapters/indices purchased from Illumina following the instructions in the Nextera guide.
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