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Old 05-08-2014, 01:56 AM   #3
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
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For 16S amplicon library prep for metagenomics studies there are few options. One is the manual you are referring from Illumina user developed protocol which is done in two steps. If you are just doing a project with no long term intention of doing similar work, following this protocol offers certainty that it is more likely to work saving time and money. It is validated, region specific primers sequences are provided and libraries can be sequenced in any Illumina system with standard Illumina sequencing reagents. Second option is designing region specific primers with Illumina adapter flow cell binding motif and barcodes which is more complicated and sequenced with custom sequencing primers. This option requires overhead expenses for synthesis of long barcoded primers and is more suitable for labs doing regular metagenomics work. Using custom primers also enables longer reads because the uninformative primers are not sequenced. Your first step should be deciding which region to amplify and sequence based on your project aims and published work. This subject including many other options has been previously discussed in this forum for instance here: http://seqanswers.com/forums/showthr...&highlight=16s
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