Microsatellite Instability

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.

You can compute Microsatellite Instability (MSI) when the DNA SNV caller is run in Tumor-Normal mode.

Microsatellite regions can be difficult to accurately map, but MSI can be inferred in silico with DRAGEN.

You can enable MSI analysis by including the --msi-command tumor-normal flag. The command executes the following steps.

1. Tabulates tumor and normal counts from the read data for each specified microsatellite region.
2. Filters out sites with insufficient coverage.
3. Determines unstable sites by performing chi-square testing. Unstable sites have repeat length distributions that are significantly shifted between tumor and normal.
4. Produces a report given an MSI score that specifies the percentage of unstable sites in every sample.

Use the following commands to compute MSI:

Command-Line Option

Description

--msi-command tumor-normal

Enables MSI.

--msi-microsatellites-file

Specify the file containing the microsatellites. You can generate this file by scanning the genome for microsatellites using an MSI-sensor. DRAGEN has tested with ≥ 10 bp homopolymers.

--msi-coverage-threshold

Specify the minimum spanning read coverage for a microsatellite. Microsatellites that do not meet the specified threshold are not included in analysis. DRAGEN recommends using 60 as the value.

The following is an example of a microsatellite file:

#chromosome location repeat_unit_length repeat_unit_binary repeat_times left_flank_binary right_flank_binary repeat_unit_bases left_flank_bases right_flank_bases

chr1 985443 1 2 15 676 992 G GGGCA TTGAA

chr1 7980985 1 0 10 231 1020 A ATGCT TTTTA

chr1 8022800 1 3 19 13 41 T AAATC AAGGC

chr1 8029500 1 2 10 39 0 G AAGCT AAAAA

chr1 9146447 1 3 15 887 248 T TCTCT ATTGA

chr1 9767837 1 3 12 704 195 T GTAAA ATAAT

The following is an example command that enables MSI analysis, including default input files.

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 \

-1 {fq1} \

-2 {fq2} \

The MSI values are output to <output prefix>.microsat_output.json.

Metric

Count

PercentageUnstableSites

18.5

TotalMicrosatelliteSitesAssessed

1581

TotalMicrosatelliteSitesUnstable

369