Sample Sheet Troubleshooting

General troubleshooting for a failed sample sheet:

In DRAGEN TruSight Oncology 500 Analysis Software, the analysis fails if a sample sheet is invalid. If an invalid sample sheet in suspected, log files can help troubleshoot a failed analysis. Use the following steps to find the log file for the sample sheet:

1. Navigate to the following location /<analysis_output>/Logs_Intermediates/SamplesheetValidation.

2. Open the SamplesheetValidation-.log file

3. Find a line with the following: SampleSheetValidationTask:NA:1 exited with return code 1 which has not been declared as a valid return code.

4. Search for errors in the sample sheet validation log and compare with the guidelines and warnings in Sample Sheet Requirements and the following tables.

Failure Type	Action
Sample Sheet not found	Verify that SampleSheet.csv is present at the top level of the run folder with the name "SampleSheet.csv". If the sample sheet is in a different location, supply the sample sheet using the --sampleSheet option
Indexes are not valid for the sequencer and/or assay	See Valid indexes for assay and instrument combinations for correct indexes for the sequencer and assay.
Pair_ID is not unique	Pair_ID column is required in the TSO500S_Data section of the sample sheet, which pairs at most one RNA and one DNA sample together for analysis. If the sample does not have a pair, use a unique pair ID for single samples.
Sample Sheet is not in v2 format	Verify that the format of the sample sheet is v2. v1 sample sheet is not compatible with DRAGEN TruSight Oncology 500 Analysis Software.
Analysis does not run	Verify the analysis starts from the run folder, and BCLs or FASTQs are in the correct locations as outlined in Starting From BCL Files and Starting From FASTQ Files respectively.

Indexes included in Library Prep Kit:

Assay	Index Set ID
TSO 500	UP1-UP16 CP1-CP16 (DNA Only)
TSO 500 HT	UDP0001–UDP0192
TSO 500 v2	UDP0001–UDP0384 (v3 index when applicable)

Troubleshooting BCL issues

Failure Type	Action
Lane Column without Values	Ensure that the column is completed. If lane is not applicable to the run, delete the column.
Format of v2 sample sheet is incorrect	Verify that the following sections and fields are present in the sample sheet and follow the individual rules in Sample Sheet Requirements [BCLConvert_Settings] - SoftwareVersion - AdapterRead1 - AdapterRead2 - AdapterBehavior - MinimumTrimmedReadLength - MaskShortReads [BCLConvert_Data] - Sample_ID - index - index2 [TS0500S_Data] - Sample_ID - Index_ID - Sample_Type - Pair_ID - Sample Feature (Optional)
HRD analysis is missing	Verify that HRD is in the Sample Feature column in the sample sheet. Refer to Sample Sheet Requirements for more information.

Troubleshooting FASTQs issues

Failure Type	Action
Sample_ID and/or Sample_Type is not present	Verify that the sample sheet has columns and values for Sample_ID and Sample_Type.
Unique sample IDs	Verify that the Sample_IDs are unique in the sample sheet.
Format of v2 Sample Sheet is incorrect	Verify that the following sections and fields are present in the sample sheet and follow the individual rules in Sample Sheet Requirements. [TS0500S_Data] - Sample_ID - Index_ID - Sample_Type - Pair_ID - Sample Feature (Optional) Verify when FASTQs were generated using the HRD add-kit (Not available in Japan), Sample Feature is added to those DNA Samples. Refer to Sample Sheet Requirements for more information.
Incorrect folder structure	Verify that the FASTQ files are in the correct structure. Refer to Starting From FASTQ Files for more information.
Invalid FASTQ input files	If the FASTQs are invalid, start TSO 500 analysis from BCL files.
HRD analysis missing	Make sure that HRD is in the correct column in the sample sheet.