Question: I'm sequencing my 10x libraries on an Illumina instrument and need a sample sheet CSV file to run BCL Convert. How can I set this up on Illumina's BaseSpace?
Answer: Below are steps you can take to create a Sample Sheet CSV in Illumina's BaseSpace Sequencing Hub (BSSH).
1) Login. First you will need to have an Illumina BaseSpace account. Here is the link where you can login:
https://basespace.illumina.com/run-planning/run-settings
After you sign in, you will come to a screen that looks like this:
2) Create a Run. To setup the run, fill out the instrument platform. If you want to complete the demultiplexing analysis on BaseSpace, the planned run can be directly accessed on the instrument. If you want to demultiplex on a separate server, you can select the 'Local' option.
3) Configuration. The next page specifies the run configuration settings. For 'Application', select the latest BCL Convert version available:
All on-market 10x Chromium and Visium products are currently available in the Illumina's BSSH Run Planner.
For 'Library Prep Kit', select the standard kit that was used from the drop-down list. The following instructions will show how to setup a Single Cell ATAC v2 sample. For guidance on how to setup another product, please refer to the the following Direct Demultiplexing pages on the 10x Genomics Support site, linked below:
- 3' Gene Expression
- 5' Immune Profiling
- Single Cell Multiome ATAC + Gene Expression
- Single Cell ATAC
- Spatial Gene Expression
- Spatial Gene Expression for FFPE
- CytAssist Spatial Gene and Protein Expression
Examples of Library Prep Kit choices:
The 'Index Adapter Kit' is the Sample Index Set plate that was used. In most cases, there is only 1 option available to select under 'Standard Kits':
4) Verify Run Configuration and Enter Sample ID. Then on the next page, the 'Library Prep Kit' and 'Index Adapter Kit' are automatically populated by your selections on the previous page. The Read Lengths and Override Cycles are also automatically populated. You do not need to change these.
The next thing to enter is your Sample ID and Index plate well information:
For ATAC/Multiome ATAC and all single index libraries, the sample index set is a blend of 4 different oligo sequences. Each oligo must be represented as a separate row in the sample sheet. For every sample, there should be 4 lines in the sample sheet. For more information, please see our direct demultiplexing page on the 10x support site.
You will see an alert indicating that the same sample has been configured with multiple indexes. This is okay/expected, and you can ignore this alert.
Please make sure to leave the AdapterRead1 and AdapterRead2 entries blank. If adapter trimming is done during demultiplexing, there is a risk of losing barcode and UMI information in downstream analysis. We generally do not recommend any adapter trimming or preprocessing FASTQ reads for any 10x product before input to our software pipelines.
You can also leave the BarcodeMismatchesIndex1 and BarcodeMismatchesIndex2 blank. This will allow the default setting of 1 mismatch.
Make sure 'gzip' is selected for FASTQ Compression Format. Then click Next to go to the next page.
5) Download Sample Sheet CSV file. From here, you can 'Export' the file to CSV format:
This will save a SampleSheet.CSV file to your local computer that looks like this:
[Header], FileFormatVersion,2 RunName,test InstrumentPlatform,NextSeq1k2k IndexOrientation,Forward [Reads] Read1Cycles,50 Read2Cycles,50 Index1Cycles,8 Index2Cycles,16 [Sequencing_Settings] LibraryPrepKits,10xATACv2 [BCLConvert_Settings] SoftwareVersion,3.10.12 CreateFastqForIndexReads,1 TrimUMI,0 OverrideCycles,Y50;I8;U16;Y50 FastqCompressionFormat,gzip [BCLConvert_Data] Sample_ID,Index,Index2 test1,AAACGGCG, test1,CCTACCAT, test1,GGCGTTTC, test1,TTGTAAGA, [Cloud_Settings] GeneratedVersion,1.11.0.202312031722 [Cloud_Data] Sample_ID,ProjectName,LibraryName,Index,Index2,LibraryPrepKitName,IndexAdapterKitName test1,,test1_AAACGGCG,AAACGGCG,,10xATACv2,10xIndexNSetA_ATACv2 test1,,test1_CCTACCAT,CCTACCAT,,10xATACv2,10xIndexNSetA_ATACv2 test1,,test1_GGCGTTTC,GGCGTTTC,,10xATACv2,10xIndexNSetA_ATACv2 test1,,test1_TTGTAAGA,TTGTAAGA,,10xATACv2,10xIndexNSetA_ATACv2
6) Use the Sample Sheet CSV file to run BCL Convert. This CSV can be used as input to run BCL Convert and generate FASTQ files:
bcl-convert --bcl-input-directory /path/to/bcl/run/directory \ --output-directory /path/to/output/directory \ --sample-sheet SampleSheet.csv
For guidance on how to run BCL Convert to generate FASTQ files, please refer to the the following Direct Demultiplexing pages on the 10x Genomics Support site for each product, linked below:
- 3' Gene Expression
- 5' Immune Profiling
- Single Cell Multiome ATAC + Gene Expression
- Single Cell ATAC
- Spatial Gene Expression
- Spatial Gene Expression for FFPE
- CytAssist Spatial Gene and Protein Expression
Although we find Illumina's BSSH Run Planner to be incredibly useful, it was developed by Illumina so we are not able to fully support all aspects of the tool or assist with setting up an instrument run. Fortunately, it is fully supported by Illumina. For further questions, issues, or feedback please contact Illumina at techsupport@illumina.com.