# How to run the DRAGEN MSI Pipeline
Microsatellites are genomic regions of short DNA motifs that are repeated 5-50 times and are associated with high mutation rates. Microsatellite Instability (MSI) results from deficiencies in the DNA mismatch repair pathway and can be used as a critical biomarker to predict immunotherapy responses in multiple tumor types.
DRAGEN MSI can work in 3 different modes determined by enabling the option --msi-command
1. Collect Evidence Mode (collect-evidence)
2. Tumor Normal Mode (tumor-normal)
3. Tumor Only Mode (tumor-only)
A **Microsatellites site** file that lists microsatellite sites of interest in a **given reference genome** is required to run this pipeline. The recommended tool for generating this file is **msi-sensor**; Information about this tool can be found [here](https://github.com/xjtu-omics/msisensor-pro/wiki).
The [scan](https://github.com/xjtu-omics/msisensor-pro/wiki/Key-Commands#scan-from-msisensor) command can be used to generate the microsatellites sites file.
msisensor-pro scan -d hg38.fa -o hg38\_microsatellites\_pro.tsv -p 1
options:
-d \[string] reference genome sequences file, \\\*.fasta format
-o \[string] output homopolymers and microsatellites file
-l \[int] minimal homopolymer size, default=5
-c \[int] context length, default=5
-m \[int] maximal homopolymer size, default=50
-s \[int] maximal length of microsate, default=5
-r \[int] minimal repeat times of microsate, default=3
-p \[int] output homopolymer only, 0: no; 1: yes, default=0
-h help
The **'-p 1'** flag is **required** to be added to the command to output only **homopolymers**; The MSI pipeline has only been **benchmarked** for **hompolymers** and **cannot** work with **repeat regions of size > 100bp**. Adding this flag should result in a compatible MSI sites file.
For **WGS and WES** samples, the **tumor-normal mode** is **recommended**. Here is an example command for a tumor-normal analysis.
dragen \\
\--msi-command tumor-normal \\
\--msi-coverage-threshold 60 \\
\--msi-microsatellites-file msi\_file \\
\--output-directory={output\_directory} \\
\--output-file-prefix={prefix} \\
\--enable-map-align=true \\
\--RGID=read\_group\_ID \\
\--RGSM=read\_group\_sample \\
\--ref-dir={reference\_directory} \\
\--enable-map-align-output=true \\
\--enable-sort=true \\
\--enable-duplicate-marking=true \\
\--tumor-fastq1 {tumor\_fq1} \\
\--tumor-fastq2 {tumor\_fq2} \\
\--fastq-file1 {fq1} \\
\--fastq-file2 {fq2}
The **tumor-only** mode requires a **panel of normals**; this can be generated by running the MSI pipeline in the collect-evidence mode. The panel of normals is required to contain **at least 20 normal** **samples** (hard-coded requirement for running the tumor-only mode).
/dragen -f \\
\--ref-dir={reference\_directory} \\
\--fastq-file1 {fq1} \\
\--fastq-file2 {fq2} \\
\--output-directory={output\_directory} \\
\--enable-map-align=true \\
\--RGID=read\_group\_ID \\
\--RGSM=read\_group\_sample \\
\--output-file-prefix={prefix} \\
\--enable-map-align-output=true \\
\--enable-sort=true \\
\--enable-duplicate-marking=true \\
\--msi-command collect-evidence \\
\--msi-coverage-threshold 60 \\
\--msi-microsatellites-file msi\_file
Once this is done, the MSI **.dist files** can be moved to a separate folder (normal\_reference\_directory) and be used as a part of the MSI pipeline in the tumor-only mode.
dragen \\
\--msi-command tumor-only \\
\--msi-coverage-threshold 60 \\
\--msi-microsatellites-file msi\_file \\
\--msi-ref-normal-dir normal\_reference\_directory \\
\--output-directory={output\_directory} \\
\--output-file-prefix={prefix} \\
\--enable-map-align=true \\
\--RGID=read\_group\_ID \\
\--RGSM=read\_group\_sample \\
\--ref-dir={reference\_directory} \\
\--enable-map-align-output=true \\
\--enable-sort=true \\
\--enable-duplicate-marking=true \\
\--tumor-fastq1 {tumor\_fq1} \\
\--tumor-fastq2 {tumor\_fq2}
More information about the MSI pipeline can be found in the [user guide](https://support-docs.illumina.com/SW/DRAGEN_v40/Content/SW/DRAGEN/Biomarkers_MSI.htm).
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