I just ran a miseq run using NEBNext Ultra DNA prep, (the bio analyzer results looked fine prior to sequencing).
We sequenced 6 samples ( 3 samples X 2 duplicates) all with different index primers (NEBNext primers for illumina 1,2,3,4,6 and 12#). However, of these six samples, three were substantially low in concentration below normalization. These three contained indices 4,6 and 12. Using the sample sheet from illumina, it said that the indices 1,2,3 would be invalid if you used them together, however for this reason we included the samples with indices 4,6 and 12 despite the low quantification. (we know we shouldve used a better combination but the miseq reagents were already thawed)
The miseq was run, and resulted in overclustering, no (0) index reads for the 6 indices, and no clustering for the second read. This resulted in single file of undetermined reads.
The questions i have are, does any one know what the most likely reason for the failure? the Overclustering? the lack of any index reads? And is there any way i can dive into the fastq file generated and see my DNA was even sequenced or if its just random stuff?
Thank you for any help!
We sequenced 6 samples ( 3 samples X 2 duplicates) all with different index primers (NEBNext primers for illumina 1,2,3,4,6 and 12#). However, of these six samples, three were substantially low in concentration below normalization. These three contained indices 4,6 and 12. Using the sample sheet from illumina, it said that the indices 1,2,3 would be invalid if you used them together, however for this reason we included the samples with indices 4,6 and 12 despite the low quantification. (we know we shouldve used a better combination but the miseq reagents were already thawed)
The miseq was run, and resulted in overclustering, no (0) index reads for the 6 indices, and no clustering for the second read. This resulted in single file of undetermined reads.
The questions i have are, does any one know what the most likely reason for the failure? the Overclustering? the lack of any index reads? And is there any way i can dive into the fastq file generated and see my DNA was even sequenced or if its just random stuff?
Thank you for any help!
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