Hi all,
I recently received the results back from a MiSeq PE-300 amplicon run, and found really weird results that we cannot explain.
We sequenced amplicons of two eukaryotic marker genes originating from soil samples. Both markers were separately PCR'd and subsequently pooled, while we added technical replicates of three of these samples together with a mock community made from pooled DNA (also replicated 3 times). We received a sufficient amount of reads per samples, but upon closer inspection, the quality in some samples dropped after around 100bp to <phred 5. We found this in the forward as well as the reverse reads. This quality drop seemed to be associated with the primer pair of just one of the two marker genes, which in bad samples was overrepresented (excluding the other gene). Notably, in the technical replicates and mock communities, we see that some replicates are good, while others are very bad...
In a second remark: the bad quality amplicons match nothing with BLAST searches, and seem to have no resemblance to the targeted gene. Yet the primers picking up non-target genes seems unlikely to us since both primers are present in the amplicon, and the Bioanalyzer results of the initial PCR products show fragments of the correct length...
The feed-back we received from the company is that the run itself went good, with sufficient clusterdensity...
The only thing that went wrong is that the Nextera kit prepared library went passed its expiry date in the sequencing company (they let us wait for 2 months).
Does anyone have any ideas what could have happened? Could these weird results be explained by the expiry of the prepared library? Thanks in advance!
I recently received the results back from a MiSeq PE-300 amplicon run, and found really weird results that we cannot explain.
We sequenced amplicons of two eukaryotic marker genes originating from soil samples. Both markers were separately PCR'd and subsequently pooled, while we added technical replicates of three of these samples together with a mock community made from pooled DNA (also replicated 3 times). We received a sufficient amount of reads per samples, but upon closer inspection, the quality in some samples dropped after around 100bp to <phred 5. We found this in the forward as well as the reverse reads. This quality drop seemed to be associated with the primer pair of just one of the two marker genes, which in bad samples was overrepresented (excluding the other gene). Notably, in the technical replicates and mock communities, we see that some replicates are good, while others are very bad...
In a second remark: the bad quality amplicons match nothing with BLAST searches, and seem to have no resemblance to the targeted gene. Yet the primers picking up non-target genes seems unlikely to us since both primers are present in the amplicon, and the Bioanalyzer results of the initial PCR products show fragments of the correct length...
The feed-back we received from the company is that the run itself went good, with sufficient clusterdensity...
The only thing that went wrong is that the Nextera kit prepared library went passed its expiry date in the sequencing company (they let us wait for 2 months).
Does anyone have any ideas what could have happened? Could these weird results be explained by the expiry of the prepared library? Thanks in advance!
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