# "Please provide the reference for the variable" error when running Maaslin2

**URL:** <https://forum.biobakery.org/t/please-provide-the-reference-for-the-variable-error-when-running-maaslin2/5272>\
**Category:** MaAsLin\
**Created:** [May 12, 2023, 2:57pm UTC](https://forum.biobakery.org/t/please-provide-the-reference-for-the-variable-error-when-running-maaslin2/5272 "2023-05-12T14:57:03Z")\
**Posts on this page:** 2\
**Page:** 1

<div class="post-metadata">

**Author:** ![paulinemaligne](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/paulinemaligne/32/2138_2.png) [@paulinemaligne](https://forum.biobakery.org/u/paulinemaligne)\
**Post date:** [May 12, 2023, 2:57pm UTC](https://forum.biobakery.org/t/please-provide-the-reference-for-the-variable-error-when-running-maaslin2/5272/1 "2023-05-12T14:57:03Z")

</div>

Hello!

I am trying to run Maaslin2 with the code:

```auto
input_data = read.table(file = "4Masslin2_input.data_kos.taxonomy.archaea.mt.2group.tsv",
                        header = TRUE, sep = "\t")
rownames(input_data) <- input_data$Geneid_ord
input_data$Geneid_ord = NULL

metadata = read.table(file = "4Masslin2_metadata_kos.taxonomy.archaea.mt.2group.tsv",
                      header = TRUE, sep = "\t")
rownames(metadata) <- metadata$Geneid_ord
metadata$Geneid_ord = NULL

# Create the 'Ctrl' column
metadata$Ctrl <- ifelse(metadata$Diagnosis == "Ctrl", "Yes", "No")

# Create the 'PD' column
metadata$PD <- ifelse(metadata$Diagnosis == "PD", "Yes", "No")

# Create the 'iRBD' column
metadata$iRBD <- ifelse(metadata$Diagnosis == "iRBD", "Yes", "No")

reference <- unique(metadata$S)
reference <- c("Methanobrevibacter_A smithii","Methanobrevibacter_A smithii_A","Methanosphaera stadtmanae","Methanomethylophilus alvus","DTU008 sp001421185","Methanomassiliicoccus luminyensis","MX-02 sp006954405","Coprobacillus cateniformis","Methanobrevibacter_C arboriphilus_A","Methanosphaera cuniculi")

Maaslin2(input_data = input_data,
         input_metadata = metadata,
         fixed_effects = c("Ctrl", "PD", "iRBD", "S"),
         reference = reference,
         min_prevalence = 0,
         output = "test",
         transform = "LOG",
         plot_heatmap = TRUE,
         plot_scatter = TRUE,
         heatmap_first_n = 50,
         max_significance = 1)

```

Examples of my metadata and input data are below:

`metadata`:

```auto
         Diagnosis D P C O F G
K00053_1 Ctrl Archaea Methanobacteriota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobrevibacter_A
K00053_2 Ctrl Archaea Methanobacteriota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobrevibacter_A
K00053_3 Ctrl Archaea Methanobacteriota Methanobacteria Methanobacteriales Methanobacteriaceae Methanosphaera
K00053_4 Ctrl Archaea Thermoplasmatota Thermoplasmata Methanomassiliicoccales Methanomethylophilaceae Methanomethylophilus
K00053_5 PD Archaea Methanobacteriota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobrevibacter_A
K00053_6 PD Archaea Methanobacteriota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobrevibacter_A
                                      S Ctrl PD iRBD
K00053_1 Methanobrevibacter_A smithii Yes No No
K00053_2 Methanobrevibacter_A smithii_A Yes No No
K00053_3 Methanosphaera stadtmanae Yes No No
K00053_4 Methanomethylophilus alvus Yes No No
K00053_5 Methanobrevibacter_A smithii No Yes No
K00053_6 Methanobrevibacter_A smithii_A No Yes No

```

`input_data`:

```auto
                tpm
K00053_1 166.502489
K00053_2 188.409788
K00053_3 69.970092
K00053_4 2.219452
K00053_5 642.522944
K00053_6 136.308126

```

As a result I receive an error:

```auto
2023-05-11 17:25:04 INFO::Writing function arguments to log file
2023-05-11 17:25:04 INFO::Verifying options selected are valid
2023-05-11 17:25:04 INFO::Determining format of input files
2023-05-11 17:25:04 INFO::Input format is data samples as rows and metadata samples as rows
2023-05-11 17:25:04 INFO::Formula for fixed effects: expr ~ Ctrl + PD + iRBD + S
Error in Maaslin2(input_data = input_data, input_metadata = metadata, : 
  Please provide the reference for the variable 'S' which includes more than 2 levels: Methanobrevibacter_A smithii, Methanobrevibacter_A smithii_A, Methanosphaera stadtmanae, Methanomethylophilus alvus, Methanomassiliicoccus_A intestinalis, UBA71 sp905187815, DTU008 sp001421185, Methanomassiliicoccus luminyensis, MX-02 sp006954405, Coprobacillus cateniformis, Methanobrevibacter_C arboriphilus_A, Methanosphaera cuniculi, Methanobrevibacter ruminantium_A.

```

Could you please suggest a solution to the error and probably the source of it?

---

<div class="post-metadata">

**Author:** ![nearinj](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/nearinj/32/2135_2.png) [@nearinj](https://forum.biobakery.org/u/nearinj)\
**Post date:** [May 12, 2023, 4:16pm UTC](https://forum.biobakery.org/t/please-provide-the-reference-for-the-variable-error-when-running-maaslin2/5272/2 "2023-05-12T16:16:00Z")

</div>

Hi there,

It seems the variable S has multiple levels and as such you need to pick a reference variable that is to be compared against during model construction. The reference variable should take in structure:

c(“S,Methanobrevibacter\_A smithii”)

This would indicate that Methanobrevibacter\_A smithii would be the reference level/category used for the variable S.

Hope that helps!

Cheers,  
Jacob