Unexpected amplicon sizes in MinION MK1D sequencing run (16S, 18S, 28S, ITS)
Hi everyone! We are using a MinION MK1D sequencer with its corresponding software for basecalling, and EPI2ME for data interpretation. We performed an amplicon sequencing run using the native barcoding kit for metagenomics. The material was extracted using the Quick-DNA Fecal/Soil Microbe Miniprep Kit. We used 16S, 18S, 28S, and ITS molecular markers, each labeled with different barcodes but pooled into a single library, and we used the short fragment buffer (SFB).
However, during sequencing, we observed inconsistencies in the expected amplicon sizes. We are seeing fragments that are both much smaller and much larger than expected within each barcode (ranging approximately from 200 bp to 1800 bp), while our expected amplicon sizes range between 600 bp and 1500 bp. These unexpected fragments appear in varying proportions across all barcodes.
Also, when we run a metagenomics analysis on the EPI2ME platform for the 16S barcode, we get unexpected eukaryotic results working with almost every database, excepting standard-8
Has anyone else experienced this issue? Could it be a barcoding problem? Could these be nucleic acids that were not amplified during PCR? Or could it be an issue related to the extraction process?
Thank you very much in advance!
However, during sequencing, we observed inconsistencies in the expected amplicon sizes. We are seeing fragments that are both much smaller and much larger than expected within each barcode (ranging approximately from 200 bp to 1800 bp), while our expected amplicon sizes range between 600 bp and 1500 bp. These unexpected fragments appear in varying proportions across all barcodes.
Also, when we run a metagenomics analysis on the EPI2ME platform for the 16S barcode, we get unexpected eukaryotic results working with almost every database, excepting standard-8
Has anyone else experienced this issue? Could it be a barcoding problem? Could these be nucleic acids that were not amplified during PCR? Or could it be an issue related to the extraction process?
Thank you very much in advance!