Troubleshoot a sequencing run

Low loading density, low live ISPs, missing test fragments, primer-dimers, and other issues can affect sequencing runs.

Observation	Possible cause	Recommended action
Failed status for a run	It is not clear whether the sequencing run data transfer completed and you can access the sequencer.	Go to the sequencer Data Management screen to confirm complete data transfer. If you are not sure the data set was transmitted, you can re-transfer it.
	The run report was successful but you are not sure whether the data was transferred.	In Torrent Suite™ Software, under the Data tab, click Completed Runs & Reports to view the run report and ensure that the file transfer was complete. Also, check if there are any error messages, such as User Aborted on the report itself. Look for a status of Error or Pending.
	You cannot determine the cause of the failed run status.	Reanalyze the run. For more information, see Reanalyze a run.
	You cannot determine the cause of the failed run status.	Send the customer support archive to your local Field Service Engineer or Technical Support for review. For more information, see Get technical support files for a completed run.
Low loading density from Ion Chef™ Instrument runs	Too little library input into template preparation. Low enriched ISP recovery is caused by unenriched ISPs with <5% templated ISPs.	Review Qubit™ QC assay results to determine if % templated ISPs was low. If % templated ISPs was <10%, increase library input to target 15–25% templated ISPs. Requantify the library. If the library yield was below recommended, rebuild the library.
	Too much library input into template preparation.	Review Qubit™ QC assay results to determine if % templated ISPs was too high after template preparation. If library is >25% but <70% templated ISPs, rebuild the library. Decrease library input to target 10-25% templated ISPs, or continue with sequencing and expect lower throughput. If library is >70% templated ISPs, check Agilent™ 2100 Bioanalyzer™ traces for adapter dimer peak (amplicon library or Ion AmpliSeq™ library peak near 70 bp; miRNA library peak near 60 bp). Repurify (reduce) library using Agencourt™ AMPure™ XP Kit clean-up steps as outlined in appropriate user guides.
	Chip adapter placement or removal was improper.	Inspect chip adapters for any obvious defects before installation.
	Chip adapter placement or removal was improper.	The best practice for removing chip adapters for Ion S5™ chips is to squeeze the adapter ends at the same time and remove the adapter by keeping it parallel to the chip.
	Bubbles were present during chip loading or during the chip run.	Air bubbles in the reagent tubes can affect various steps through the sequencing run, including loading density. Let reagents settle before using.
Low loading density from Ion OneTouch™ 2 System runs	Too little library input into template preparation. Low enriched ISP recovery is caused by unenriched ISPs with <5% templated ISPs.	Review Qubit™ QC assay results to determine if % templated ISPs was low. If % templated ISPs was <10%, increase library input to target 15–25% templated ISPs. Requantify the library. If the library yield was below recommended, rebuild the library.
	Too much library input into template preparation.	Review Qubit™ QC assay results to determine if % templated ISPs was too high. If library is >25% but <70% templated ISPs, rebuild the library. Decrease library input to target 10-25% templated ISPs, or continue with sequencing and expect lower throughput. If library is >70% templated ISPs, check Agilent™ 2100 Bioanalyzer™ traces for adapter dimer peak (amplicon library or Ion AmpliSeq™ library peak near 70 bp; miRNA library peak near 60 bp). Repurify (reduce) library using Agencourt™ AMPure™ XP Kit clean-up steps as outlined in appropriate user guides.
	Low pre-enriched ISP recovery.	Review Qubit™ QC assay results to determine if ISP recovery was low after template preparation.
		Ensure that ISPs are properly vortexed (1 minute at maximum speed as suggested in the user guide) before the addition of amplication mix.
		Ensure proper OT2 instrument setup and performance.
	Low enriched ISP recovery.	Ensure proper reagent setup for ES enrichment.
		Ensure that enrichment beads (Dynabeads™ MyOne™ beads) are washed and resuspended in the correct washing solution.
		Properly prepare the fresh melt-off solution.
	Improper addition of sequencing primer and polymerase.	Ensure that sequencing primer and polymerase were added.
		During the primer annealing step, it is important to thoroughly resuspend the enriched ISP pellet before and after adding the sequencing primer. This helps to break up ISP clumps and ensure even sequencing primer distribution to the templates on the ISPs.
		Properly mix ISPs during the primer annealing step. Improper mixing can cause lower than expected key signal and possible misidentification of template ISPs.
	Wrong loading protocol was used.	Use the recommended protocol for the chip type and sequencing kit.
Low percentage of live ISPs. The Live (% enrichment) metric includes both live TF and library ISPs. When evaluating the percent enrichment metric, also consider the ratio of test fragment ISPs to library ISPs. If the ratio of TF ISPs is higher than expected, the reported percent of enriched library ISPs is not accurate. Instead, calculate the library enrichment from the values in the run report table: [Library ISPs/(Live ISPs - TF ISPs)] x 100 = Library ISP % Enrichment.	Improper addition of sequencing primer and polymerase.	Review Test Fragment (TF) metrics. If TFs are missing or the signal is low: primer or polymerase can have been added incorrectly.
	Improper addition of sequencing primer and polymerase.	Ensure proper addition of sequencing primer and polymerase.
	Poor library amplification onto the ISPs (template preparation).	Review Test Fragment (TF) metrics. If TFs are present and have the expected key signal and quality, check the Qubit™ QC assay. If results indicate <10% templated unenriched ISPs, then too little library was added.
		Check Ion OneTouch™ 2 System performance by downloading the log files.
	Too little library input into template preparation.	Review Test Fragment (TF) metrics. If TFs are present and have the expected key signal and quality, check the Qubit™ QC assay. If results indicate <10% templated unenriched ISPs, then too little library was added.
	Too little library input into template preparation.	Check Ion OneTouch™ 2 System performance by downloading the log files.
	Enrichment failure.	Check the ES enrichment process to ensure that fresh melt-off and the correct enrichment beads (Dynabeads™ MyOne™ beads) and solutions are used.
	Enrichment failure.	If >5-µL residual volume is left in wells 1 through 8, perform a residual volume test.
Test Fragment-1 is not present or conversion is low There are two test fragment controls: TF-C is a sequencing control and TF-1 is an amplification control. Starting in Torrent Suite™ Software 5.4, only TF-1 is present.	Poor amplification onto the ISPs (template preparation).	Review Qubit™ QC assay results to determine if % templated ISPs was low after template preparation.
	Poor amplification onto the ISPs (template preparation).	Ensure that reagents were set up correctly.
	Sequencing or consumables problem occurred.	Ensure that sequencing primer was annealed and sample was incubated with polymerase.
		Ensure that all reagents were from the same sequencing kit type. Do not swap reagents between kit types.
		Ensure that reagents set up were correctly.
		Send Customer Support Archive (CSA) to Technical Support.
Lower than expected number of live library ISPs with a key sequencing matching the library key (TCAG).	Low loading density reported.	See recommendations for low loading density.
	Bad library key used.	Ensure that the library was properly prepared. This is less likely to be the problem if an Ion library kit was used for library construction.
	Bad library key used.	Ensure that the template preparation was successful. Check Qubit QC assay results. Send Customer Support Archive (CSA) to Technical Support.
	Poor library quality or templating efficiency.	Ensure that the library was properly prepared. This is less likely to be the problem if an Ion library kit was used for library construction.
	Poor library quality or templating efficiency.	Ensure that the template preparation was successful. Check Qubit QC assay results. Send Customer Support Archive (CSA) to Technical Support.
High polyclonal count	Too much library input into template preparation.	Verify library quantification and template preparation with QC (Qubit assay). If the library key signal is lower than expected, % polyclonal estimate might be inaccurate.
High number of low quality reads. Reads are too short after quality trimming.	Poor library quality or templating efficiency: bad or low key signal.	Check Agilent™ Bioanalyzer™ Instrument trace to ensure that library is within the recommended size range (amplified, non-equalizer libraries only).
		Ensure that library was properly prepared (correct adapter sequences, adapter ligation).
		Review the library ISP QC data Qubit™ data.
High relative primer dimer count	Library preparation resulted in high number of primer dimers.	Check Agilent™ Bioanalyzer™ Instrument trace. Non-barcoded adapter dimers ~70 bp. Barcoded adapter dimers ~80 bp.
High relative primer dimer count		Repurify library to remove adapter dimers.
Low Final Library ISPs count	Low ISP loading density reported.	Review well classification and read filtering results to narrow troubleshooting focus.
Low Final Library ISPs count	Poor library read filtering reported.	Send a Customer Support Archive (CSA) to Technical Support.

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Troubleshoot a sequencing run