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Sequencing primers did not bind to the clusters in Illumina Miseq600 sequencing platf
I ran an Illumina Miseq600 sequencing and it failed - a cluster density is only 79 k/mm2 with 0% passing filter. From the thumbnail image at 5 cycles, we detected background clusters in a large number which likely indicate the cluster generation, but these clusters were not sequenced.
We, as well as a technical support, suspected that the sequencing primers did not bind to the generated clusters, thus the sequencing process was not performed and therefore a large number of background clusters were observed.
Kindly noted that I followed Caporaso et al. (2012)’s protocols (https://press.igsb.anl.gov/earthmicr...standards/its/), with modified primers for ITS2 region using universal ITS3KYO2 and ITS4 primers (Toju H. et al.,2012).
Please suggestions on how the ideal custom sequencing primers should be (i.e. degenerated bases, Tm, etc.)
Thank you
We, as well as a technical support, suspected that the sequencing primers did not bind to the generated clusters, thus the sequencing process was not performed and therefore a large number of background clusters were observed.
Kindly noted that I followed Caporaso et al. (2012)’s protocols (https://press.igsb.anl.gov/earthmicr...standards/its/), with modified primers for ITS2 region using universal ITS3KYO2 and ITS4 primers (Toju H. et al.,2012).
Please suggestions on how the ideal custom sequencing primers should be (i.e. degenerated bases, Tm, etc.)
Thank you