Hello all,
I am using the Ion 16S Metagenomic Kit (Thermofisher Scientific) on an Ion Torrent PGM platform. However, instead of 16S primer, I am using ITS primers. I want to isolate and sequence fungal DNA from 176 leaf samples. Once I generate my PCR product, I need to end-repair each PCR, then ligate adapter barcodes, and then complete nick-repair.
Can I pool all 176 samples with unique barcodes into one library and treat it as one sample to do end-repair, ligation and nick-repair procedures just once? Or do I need to treat each of my 176 PCR products separately when I do the end-repair, ligation and nick-repair?
If you want to compare he fungal communities between samples, you can't pool all the samples to use only one barcode, because after sequencing you will not be available to separate or group the sequences per sample.
Depends of what do you want to do with this samples. An also, with only one sample, you can't do any statistical analysis, you need replicates.
Hello José and Mathew,
Thank you for your comments. For clarification, yes I'll be adding the unique barcodes during the initial PCR. Additionally, I intend to use:
- 25 unique barcode sequences attached to the ITS-1F primer
- 7 unique barcodes sequences attached to the ITS-2R primer
- 1 non-barcoded ITS-2R Primer
Combinations of the 25 ITS-1F primers with 8 ITS-2R primers should allow 200 unique barcode combinations, which exceeds my 176 samples.
Cheers,
Gary
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