Demultiplexing Multisample Tapestri Runs

With the release of sample multiplexing, customers are able to multiplex up to 3 samples on one Tapestri run using either antibody hashing or genotype multiplexing with seamless demultiplexing integrated into the following product lines:

• Antibody hashing is currently compatible with Tapestri DNA and Tapestri GE DNA pipelines.
• Genotype multiplexing is currently compatible with Tapestri DNA and Tapestri DNA + Protein pipelines.

Tapestri Pipeline v3.4 application can be used to demultiplex each cell to its original sample. This article describes the method to process your multisample (multiplexed) GE DNA, Tapestri DNA or Tapestri DNA + Protein pipeline runs.

• Tapestri DNA with Genotype Demultiplexing
• Tapestri DNA+Protein with Genotype Demultiplexing
• Tapestri DNA with Antibody Hashing
• Tapestri GE DNA with Antibody Hashing

Tapestri DNA / DNA+Protein Genotype Demultiplexing

Genotype multiplexing is currently compatible with Tapestri DNA and Tapestri DNA + Protein pipelines. To demultiplex such runs the user needs to provide germline variant information for all the samples multiplexed in a run. The genotype demultiplexing pipeline is a three step process:

1. Process Bulk NGS data (optional) - A user can choose to run Mission Bio's Bulk NGS kit to process each single sample independently before multiplexing it. The result of the Bulk pipeline can be used to generated a germline variant file.
2. Create Germline Variant CSV File - To generate the sample variants CSV file use the "Merge Bulk Runs" pipeline on the Bulk NGS runs or create the CSV file manually by providing the list of variants which are distinct across the multiplexed samples.
3. Process the multisample run - Use the sample variants file created in the previous step to demultiplex the multisample run and obtain the sample level output files.

Step 1 - Process Bulk NGS Runs (optional)

The first step of the Genotype Demultiplexing pipeline is to run the Bulk NGS data processed using Mission Bio's NGS kits. Start the DNA v3.4 pipeline with "Bulk" selected in Run Mode dropdown. Choose the relevant genome, panels and FASTQ files and start the run:

Step 2 - Create Sample Variants CSV File 

There are two options to create the sample variants file - (i) Merge the bulk runs from the previous step, (ii) Manually create a csv file in the correct format using pre-existing germline variant data. To create the file manually follow the instructions defined in this article. To generate the file using the Merge Bulk Runs pipeline follow the steps given below:

1. Prerequisites - Bulk NGS runs as defined in the Step1 are all successfully complete with one cell.
2. Click Start run and add the run name.
3. Select the pipeline "Merge Bulk Runs v3.4".
4. Click the button Add to select the Bulk H5 files.
5. Select bulk h5 files for the sample that would be multiplexed in a single Tapestri run.
6. Add the unique Sample Name. Please note the sample name should be unique, at least 3 character long and alphanumeric. It should not contain any special character other than "-" or "_".
7. Click Submit.

This run takes ~10-12 minutes to complete, once it is successfully complete open the run and check the variant report file. This report provide a summary of the number of germline variants detected and performance metrics for each. It also highlights if the samples are suitable to be multiplexed in a single run. If there are not enough differentiating variants then that combination of samples should not be multiplexed.

Once this run completes and there are sufficient variants to be used for demultiplexing move to the next step.

Step 3 - Process Multisample Run

This is the final step in the process of Genotype Demultiplexing workflow. Once the sample variants CSV file is available either from external sources or the Merge Bulk Run pipeline, start the demultiplexing run following the steps given below:

1. Click Start Run and add the Run name.
2. On the Next step, select the pipeline DNA v3.4 or DNA + Protein v3.4.
3. Choose the Genotype Demultiplexing as the Run Mode from the dropdown.
4. From the Sample Variants File dropdown, select the germline truth file generated from the previous run (Pipeline Generated) or upload your own file (User Uploaded).
5. Select the Genome, Panel and FASTQ files.
6. Start the run.

Once the run is completed the demultiplexed samples files will be available on the Output tab for the run. For more details on the output files read this article.

Tapestri DNA Antibody Hashing

Tapestri DNA Antibody Hashing runs are processed using DNA + Protein pipeline with Antibody Demultiplexing as Run Mode. Unlike Genotype Demultiplexing, this pipeline is a single step process. It requires a Demultiplexing Protein panel which can be created by following the steps in this article. Once the panel is ready follow the step below to start the pipeline:

1. Click Start Run and add the run name.
2. Select the pipeline DNA + Protein.
3. Select Antibody Demultiplexing in the Run Mode dropdown.
4. Select the Genome, DNA panel, Demultiplexing Protein panel and the FASTQ file.
5. Start the run.

Once the run completed the demultiplexed samples files will be available on the Output tab for the run. For more details on the output files read this article.

Tapestri GE DNA Antibody Hashing

Tapestri GE DNA Antibody Hashing runs are processed using GE DNA + Protein pipeline. Unlike Tapestri DNA Antibody Hashing, there is no Run Mode for GE and it will run the demultiplexing step if the protein panel contains the column "Sample_ID". To create the Protein panel with the demultiplexing information follow the steps in this article. Once the panel is ready follow the step below to start the pipeline:

1. Click Start Run and add the run name.
2. Select the pipeline GE DNA + Protein.
3. Select the Genome, Report Type, GE DNA panel, Protein panel and the FASTQ file.
4. Start the run.

Once the run completed the demultiplexed samples files will be available on the Output tab for the run.