Troubleshooting Using Control Libraries; Troubleshooting Using Control Ion Sphere ™ Particles - Thermo Scientific Ion GeneStudio S5 User Manual

Troubleshooting using control libraries

Observation
Ion Sphere Test Fragments
™
are not present in the Test
Fragment Report section of the
run report, and library
sequencing is poor
Control Ion Sphere Particles
™
are present in the run report,
but AQ20 throughput is poor
Troubleshooting using Control Ion Sphere
Ion GeneStudio S5 Instrument User Guide
Possible cause
• Chip loading was poor.
• Control Ion Sphere
Particles were not added
to the sample
• The quality of the library
was poor.
• The quality of the template
preparation was poor.
To prepare Control Ion Sphere
an installation or troubleshooting sequencing run:
1. Create a Planned Run.
2. Clean and initialize the Ion GeneStudio S5 Sequencer.
3. Prepare the Control Ion Sphere
a. Vortex the control ISPs for 5 seconds, then centrifuge for 2 seconds before
taking aliquots.
b. Add 66 µL of control ISPs to an empty 0.2-mL PCR tube (non-polystyrene).
c. Add 150 µL of Ion S5
4. Anneal the sequencing primer to the enriched ISPs, then follow the remaining
procedures in Ion 540
chip and start the sequencing run.
Troubleshooting using control libraries
1. Confirm that the Control Ion Sphere
Particles (included in the Ion S5
™
Installation Kit) were added.
2. If controls were added, contact Technical
Support.
Verify the quality of the library and template
preparations using quality assessment
procedures recommended in the appropriate
library and template preparation user guides.
1. Use the Human CEPH Genomic DNA
Control or Human CEPH Control 200
Library, included in the Ion S5
Kit (Cat. No. A27760), to prepare
template-positive ISPs with the
Ion OneTouch
Ion Chef
2. Use the ISPs in an Ion GeneStudio S5 run.
3. If AQ20 throughput is still below
specification, verify the quality of
unenriched and enriched ISPs to identify
a problem in template preparation.
4. If ISP quality is good, but AQ20 throughput
is below specification, contact Technical
Support.
™ Particles
™
Particles, provided in the Ion S5
Particles for sequencing:
™
Annealing Buffer to the tube.
™
Kit – OT2 User Guide (Pub. No. MAN0010850) to load a
™
Appendix B Troubleshooting
Recommended action
™
Controls
™
2 Instrument, or the
™
Instrument.
™
™
Installation Kit, for
B
™
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