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/earthmicrobiome/protocols-and-standards/its/), with modified primers for ITS2 region using universal ITS3KYO2 and ITS4 primers (Toju H. et al.,2012). Please find below the details of my amplicon primers and sequencing primers.
If you are doing amplicon sequencing on the Miseq platform, you might need to add PhiX control DNA or, alternatively, use a mixture of adapter primers with a variable number of arbitrary bases between the sequencing primer site and the gene-specific amplification site to create a stagger effect. The reason that these are necessary is that, if all your amplicons start with the same stretch of bases, the instrument has trouble focusing its optics and this can lead to miscalled clusters.
Another issue is that your adapter primers need to be very high quality. They should be PAGE or HPLC purified so that the fidelity of your fragments (particularly at the 5' end) is good enough for them to properly bind the flow cell and/or sequencing primers.
Possible reasons could include primer variation and single nucleotide polymorphisms. Did you check your primer specificity in ncbi?
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