Workflow Requirements
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Supports assays prepared with the TruSight Oncology UMI Reagents. |
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Compatible with sequencing on the HiSeq 2500/4000. Compatibility with the NovaSeq 6000 was demonstrated but not extensively tested. |
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Sequencing settings are as follows. |
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Recommended sequencing runs are the following. |
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2 × 125 base pairs on the HiSeq 2500 with high output run mode |
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2 × 151 base pairs on the HiSeq 4000 or NovaSeq 6000 |
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Connectivity to send sequencing run output data to BaseSpace Sequence Hub. Streaming run data directly to BaseSpace from the sequencing system is recommended. |
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HiSeq 2500 System User Guide (15035786) |
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HiSeq 4000 System Guide (15066496) |
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NovaSeq 6000 Sequencing System Guide (1000000019358) |
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Sequencing Run Setup is as follows. |
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The following UMI parameters must be included in the sample sheet under the [Settings] section (Parameter, Value). |
CAUTION
Using biosamples containing aggregated data from multiple runs has not been fully tested. Using single run biosamples is recommended.
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Local FASTQ file generation |
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UMI parameters must be in the sample sheet during local bcl2fastq, otherwise FASTQ files will not be compatible with the UMI Error Correction BaseSpace Sequence Hub App. |
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For an easier transition between the UMI Error Correction BaseSpace Sequence Hub App and the UMI Error Correction Local App (without file renaming), do not use underscores in Sample IDs and leave the Sample Name blank in the sample sheet. |
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Command line settings for bcl2fastq in BaseSpace Sequence Hub are as follows. |
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--minimum-trimmed-read-length 35 |
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--mask-short-adapter-reads 35 |
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--adapter-stringency 0.9 |
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--ignore-missing-filter |
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--ignore-missing-positions |
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--ignore-missing-controls |
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--auto-set-to-zero-barcode-mismatches |
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--find-adapters-with-sliding-window |