I'm doing amplification of ITS2 region for fungal sequencing on Miseq. At times, 2 or three bands appeared on gel electrophoresis (range between 400-600 bp). Should all the appeared bands in this range be used for sequencing or only a selected band?
(Note: We used custom designed barcoded primers to amplify ITS2 region following the protocol from Caporaso Illumina sequencing (we prepared library prep manually not kit). So, we are concern whether the bands formed are really fungal barcoded amplicons not amplicon with no barcode)
Hi, I would sequence all of them. What is not clear is if you are working with an isolate or an environmental sample. Either way the ITS2 can range from 14 to over 700 bases.
Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing Yang RH, Su JH, Shang JJ, Wu YY, Li Y, et al. (2018) Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing. PLOS ONE 13(10): e0206428. https://doi.org/10.1371/journal.pone.0206428
- Badges
- Science method