Hello everyone,
I am the current manager for the Marine Gene Probe Lab (MGPL) at Dalhousie University and we have been trying to sequence eDNA samples from freshwater lakes using the 12S MiFish-U primers from Miya et al. (2015). The lab has an in-house MiSeq for sequencing with v2 chemistry. Our protocol differs from the original paper and, while it gets some usable data, is often resulting in poor R2 quality and merge rates. We have also been having consistent issues with over-clustering, even when the libraries were diluted to 6pM prior to loading. I figure this is at least part of the reason for the poor R2 reads. We've also been getting higher amounts of adapter in our sequencing reads than we would like to see, with some instances taking up about 50% of the reads for a sample. I suspect those instances are due to poor template quality or insufficient template.
The actual preparation of the libraries is done by me (our Research Associate processes the data), and I'm wondering if anyone else on here is attempting similar experiments or has successfully generated good quality data from eDNA template. It's proving tricky for us. Thanks!
I am the current manager for the Marine Gene Probe Lab (MGPL) at Dalhousie University and we have been trying to sequence eDNA samples from freshwater lakes using the 12S MiFish-U primers from Miya et al. (2015). The lab has an in-house MiSeq for sequencing with v2 chemistry. Our protocol differs from the original paper and, while it gets some usable data, is often resulting in poor R2 quality and merge rates. We have also been having consistent issues with over-clustering, even when the libraries were diluted to 6pM prior to loading. I figure this is at least part of the reason for the poor R2 reads. We've also been getting higher amounts of adapter in our sequencing reads than we would like to see, with some instances taking up about 50% of the reads for a sample. I suspect those instances are due to poor template quality or insufficient template.
The actual preparation of the libraries is done by me (our Research Associate processes the data), and I'm wondering if anyone else on here is attempting similar experiments or has successfully generated good quality data from eDNA template. It's proving tricky for us. Thanks!
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