# 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 `//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](https://help.connected.illumina.com/tso500/dragen-tso-500-guides/dragen-tso-500-v2.6/run-planning/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 | | | TSO 500 HT | | | TSO 500 v2 | | ## 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](https://help.connected.illumina.com/tso500/dragen-tso-500-guides/dragen-tso-500-v2.6/run-planning/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](https://help.connected.illumina.com/tso500/dragen-tso-500-guides/launching-analysis/run-dragen-tso-software/command-line-options#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. |