We were to sequence 81 samples in the last Miseq run, but a last minute mistake made us to not apply a few. So the final pool had 73 samples. However, we did not deleted the names of these samples from the SampleSheet which means Miseq “didn’t know” they were out. They run like fake samples. We expected they had no reads, of course. But now I got the fastq and surprisingly found that there are a few reads from within those fake samples.
Negative-reaction-control_S82_R1.fastq.gz 34 Negative-extraction-control-1_S30_R1.fastq.gz 35 IG19-202_S2_R1.fastq.gz 37 not run IG19-233_S21_R1.fastq.gz 39 not run IG19-234_S22_R1.fastq.gz 41 not run IG19-208_S8_R1.fastq.gz 44 not run IG19-235_S23_R1.fastq.gz 48 not run Negative-extraction-control-2_S81_R1.fastq.gz 80 IG19-175_S50_R1.fastq.gz 85 not run IG19-174_S49_R1.fastq.gz 110 not run IG19-176_S51_R1.fastq.gz 140 not run IG19-196_S66_R1.fastq.gz 1713 IG19-200_S70_R1.fastq.gz 5184 IG19-195_S65_R1.fastq.gz 5235 IG19-199_S69_R1.fastq.gz 5346 IG19-198_S68_R1.fastq.gz 5387 ... Positive-control_S80_R1.fastq.gz 17621 Undetermined.fastq.gz 318405 --Total number of reads on the run = 2540687
Does someone had any experience or clue on why that happened?
I am on two hypothesis:
- barcode cross-talk even within samples that really don’t exist. Somehow the machine found those reads belonged to those samples…
- contamination issues. Even the samples were not real their read numbers are close to those from the negative controls. Would that indicate there is actually (maybe also) a contamination around? But that would mean too that 34, 35 and 80 reads within ~2.5 million would be interpreted as significant, which I really don’t know.