Help needed for Zero Passed-filter reads on MiSeq run

[NGS777]

Hello,

We created a libary by digesting genomes with restriction enzymes and putting adapters. The BioAnalyzer trace looked ok. We added 30% Phix controls and then ran a sequencing on MiSeq. There are 25M Clusters generated with 1133K/mm density, but there were absolutely zero passed-filter reads and were zero yiled. The run finished without error and BaseSpace has indicated that it is a failed run.


We are just wondering what caused the failure;

1. Library construction ?
2. Library input ?
3. Mechanical ?
4. Reagents ?
5. Software ?
6. etc

We have contacted with Tech supports, but we also want help and any comments from you.

Thank you!

[microgirl123]

Have you looked at your tile images in SAV? That may give you some clue as to what happened.

[NGS777]

Thank you for comment!

I found tile images from the first cyles, but unfortunatley, I don't know how to look at them. Could you comment on the images whether they look normal or not?

Thank you in advance!

[BBthekid007]

To my eye, those thumbnails show a run that's heavily overclustered. Although the MiSeq calculates a density of 1133k/mm, many of the 'clusters' it's calling may actually be multiple overlapping clusters. The mixed base information that would come out of such overlapping clusters would cause the cluster to fail to pass filter, which appears to be exactly what happened. Maybe try loading less of your library?