Contents

1 Why strainspy?

strainspy fits strain-level association models from ANI/abundance profiles and metadata. This guide follows the README workflow and is written as a practical first analysis.

You will walk through the core path most users care about:

  1. Load metadata and ANI profiles.
  2. Filter to informative strains.
  3. Fit a zero-inflated beta model with glmZiBFit().
  4. Rank and interpret hits with top_hits().
  5. Explore taxonomy-aware summaries and plot-ready outputs.
  6. Prepare data for downstream prediction and diversity analyses.

All examples use bundled data so the workflow runs end-to-end.

2 Installation

Install the release version from Bioconductor:

if (!requireNamespace("BiocManager", quietly = TRUE)) {
    install.packages("BiocManager")
}
BiocManager::install("strainspy")

Conda, container and GitHub installation options are described in the package README.

3 Load package and example data

The bundled files are described in inst/extdata/README.md (system.file("extdata", "README.md", package = "strainspy")).

library(strainspy)

meta_path <- system.file("extdata", "example_metadata.csv.gz", package = "strainspy")
sylph_path <- system.file("extdata", "example_sylph_profile.tsv.gz", package = "strainspy")
taxonomy_path <- system.file("extdata", "example_taxonomy.tsv.gz", package = "strainspy")

meta <- read.csv(meta_path)
meta$Case_status <- factor(meta$Case_status)

taxonomy <- read_taxonomy(taxonomy_path)
se <- read_sylph(sylph_path, meta_data = meta)

# Recommended default: keep strains observed in at least 10% of samples
min_nonzero <- ceiling(0.10 * ncol(se))
se <- filter_by_presence(se, min_nonzero = min_nonzero)

cat("Samples:", ncol(se), "\n")
## Samples: 200
cat("Retained strains:", nrow(se), "\n")
## Retained strains: 634
cat("min_nonzero used:", min_nonzero, "\n")
## min_nonzero used: 20

4 Choosing key wrapper inputs

If you use the high-level strainspy() wrapper, three inputs should be chosen deliberately:

  1. design_formula: the predictors you want to test.
  2. coef: which coefficient index from that design to report in top_hits().
  3. min_nonzero: prevalence threshold (recommended default: ceiling(0.10 * n_samples)).

For the design ~ Case_status + Age_at_collection, coef = 2 targets Case_status. You can verify term order with colnames(model.matrix(~ Case_status + Age_at_collection, meta)).

5 Fit the main model (glmZiBFit)

design <- as.formula("~ Case_status + Age_at_collection")

fit <- glmZiBFit(
  se,
  design,
  nthreads = 2L,
  return_vcov = FALSE
)
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fit
## strainspy_fit object
## ------------------------------
## Design formula: ~Case_status + Age_at_collection
## ------------------------------
## Converged=634, Failed=0
## ------------------------------
## Coefficients:  634  features x  3  terms
## ------------------------------
## ZI Coefficients:  634  features x  3  terms
## ------------------------------
## Residuals available:  Yes 
## Call:
## glmZiBFit(se = se, design = design, nthreads = 2L, return_vcov = FALSE)
## ------------------------------
## Priors used:
## strainspy_priors object
## Method:  preset_weak 
## 
## Priors:
##  - fixef : 2 coefficients
##  - fixef_zi : 2 coefficients
## 
## Example priors:
##         prior    class              coef
## 1 normal(0,5)    fixef     Case_statusPD
## 2 normal(0,5)    fixef Age_at_collection
## 3 normal(0,5) fixef_zi     Case_statusPD
## 4 normal(0,5) fixef_zi Age_at_collection
## 
## Bootstrap summary for fixef priors:
##    Min. 1st Qu.  Median    Mean 3rd Qu.    Max. 
##      NA      NA      NA     NaN      NA      NA 
## 
## Bootstrap summary for fixef_zi priors:
##    Min. 1st Qu.  Median    Mean 3rd Qu.    Max. 
##      NA      NA      NA     NaN      NA      NA 
## ------------------------------

6 Find top hits

top_hits() is usually the first table you inspect after fitting. It highlights contigs with the strongest phenotype association and provides adjusted p-values.

hits <- top_hits(fit, coef = 2, alpha = 1)
head(hits[, c("Contig_name", "coefficient", "p_value", "p_adjust")])
## # A tibble: 6 × 4
##   Contig_name                                       coefficient p_value p_adjust
##   <chr>                                                   <dbl>   <dbl>    <dbl>
## 1 NZ_WSNW01000023.1 Coprobacillus cateniformis str…       0.167 1.80e-1   1     
## 2 UREB01000001.1 uncultured Clostridiales bacteriu…       0.248 1.01e-4   0.0638
## 3 DVOB01000010.1 MAG TPA_asm: Candidatus Copromorp…       0.231 1.08e-4   0.0681
## 4 JAHZEH010000001.1 MAG: Clostridia bacterium isol…       0.350 1.40e-4   0.0882
## 5 NZ_JXXK01000001.1 Ruthenibacterium lactatiforman…       0.121 1.41e-4   0.0887
## 6 NZ_VICC01000001.1 Actinomyces oris strain CCUG 3…       0.111 1.43e-1   1

7 Add taxonomy context

When taxonomy is available, hadjust() summarizes significance across taxonomic levels.

hier_hits <- hadjust(fit, coef = 2, taxonomy = taxonomy)
head(hier_hits[, c("Level", "Model", "Name", "p_adjust")])
## # A tibble: 6 × 4
##   Level  Model         Name               p_adjust
##   <chr>  <chr>         <chr>                 <dbl>
## 1 Phylum Beta          Bacillota_A          0.0151
## 2 Class  Beta          Clostridia           0.0151
## 3 Order  Beta          Christensenellales   0.0320
## 4 Phylum Zero-Inflated Bacillota_I          0.0391
## 5 Class  Zero-Inflated Bacilli_A            0.0391
## 6 Order  Zero-Inflated Erysipelotrichales   0.0391

8 Generate plot-ready outputs

The README demonstrates visualization early because it helps with interpretation. The two main plots are rendered below. Set plot = FALSE in either call to get the underlying table instead of the figure.

plot_volcano(fit, coef = 2)

plot_manhattan(fit, coef = 2, taxonomy = taxonomy)

9 Diversity and prediction preparation

Beyond hit calling, strainspy supports ecological summaries and model-ready tables.

rich <- estimate_sample_richness(se, taxonomy)
beta_dist <- estimate_beta_diversity(se, distance_only = TRUE)

pred_df <- prep_for_prediction(
  sy = se,
  outcome = "Case_status",
  contigs = rownames(se)[1:3],
  covariates = "Age_at_collection",
  use_genome_names = FALSE
)

length(rich$species_richness)
## [1] 200
class(beta_dist)
## [1] "dist"
dim(pred_df)
## [1] 200   5

10 Working with other profile inputs

The same downstream workflow can be used with other supported profile readers.

example_sourmash_path <- system.file("extdata", "example_sourmash.csv.gz", package = "strainspy")
example_metaphlan_path <- system.file("extdata", "metaphlan_merged.tsv.gz", package = "strainspy")

sm <- read_sourmash(example_sourmash_path, meta)
mp <- read_metaphlan(example_metaphlan_path, meta)

class(sm)
## [1] "SummarizedExperiment"
## attr(,"package")
## [1] "SummarizedExperiment"
class(mp)
## [1] "SummarizedExperiment"
## attr(,"package")
## [1] "SummarizedExperiment"

11 Notes

12 Next steps

After this walkthrough, common next analyses are:

  1. Tighten alpha in top_hits() to focus on the strongest hits.
  2. Use taxonomy-informed Manhattan summaries to prioritize clades.
  3. Export prep_for_prediction() outputs to train external classifiers.

13 Session information

sessionInfo()
## R version 4.6.1 Patched (2026-06-24 r90190)
## Platform: x86_64-apple-darwin20
## Running under: macOS Ventura 13.7.8
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.6-x86_64/Resources/lib/libRblas.0.dylib 
## LAPACK: /Library/Frameworks/R.framework/Versions/4.6-x86_64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.1
## 
## locale:
## [1] C/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## time zone: America/New_York
## tzcode source: internal
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] strainspy_0.99.3 BiocStyle_2.41.0
## 
## loaded via a namespace (and not attached):
##   [1] RColorBrewer_1.1-3          jsonlite_2.0.0             
##   [3] magrittr_2.0.5              magick_2.9.1               
##   [5] TH.data_1.1-5               estimability_2.0.0         
##   [7] farver_2.1.2                nloptr_2.2.1               
##   [9] rmarkdown_2.32              fs_2.1.0                   
##  [11] vctrs_0.7.3                 minqa_1.2.8                
##  [13] ggtree_4.3.0                tinytex_0.61               
##  [15] htmltools_0.5.9             S4Arrays_1.13.2            
##  [17] SparseArray_1.13.4          gridGraphics_0.5-1         
##  [19] sass_0.4.10                 bslib_0.12.0               
##  [21] htmlwidgets_1.6.4           sandwich_3.1-3             
##  [23] emmeans_2.0.4               zoo_1.9-1                  
##  [25] cachem_1.1.0                TMB_1.9.25                 
##  [27] lifecycle_1.0.5             pkgconfig_2.0.3            
##  [29] Matrix_1.7-6                R6_2.6.1                   
##  [31] fastmap_1.2.0               rbibutils_2.4.1            
##  [33] MatrixGenerics_1.25.0       digest_0.6.39              
##  [35] numDeriv_2016.8-1.1         aplot_0.3.2                
##  [37] patchwork_1.3.2             S4Vectors_0.51.10          
##  [39] GenomicRanges_1.65.4        labeling_0.4.3             
##  [41] polyclip_1.10-7             abind_1.4-8                
##  [43] mgcv_1.9-4                  compiler_4.6.1             
##  [45] fontquiver_0.2.1            withr_3.0.3                
##  [47] S7_0.2.2                    BiocParallel_1.47.0        
##  [49] viridis_0.6.5               ggforce_0.5.0              
##  [51] R.utils_2.13.0              MASS_7.3-66                
##  [53] rappdirs_0.3.4              DelayedArray_0.39.8        
##  [55] harmonicmeanp_3.0.1         tools_4.6.1                
##  [57] otel_0.2.0                  ape_5.8-1                  
##  [59] R.oo_1.27.1                 glue_1.8.1                 
##  [61] nlme_3.1-171                grid_4.6.1                 
##  [63] generics_0.1.4              snow_0.4-4                 
##  [65] glmmTMB_1.1.15.2            gtable_0.3.6               
##  [67] R.methodsS3_1.8.2           tidyr_1.3.2                
##  [69] data.table_1.18.6.1         utf8_1.2.6                 
##  [71] XVector_0.53.0              BiocGenerics_0.59.12       
##  [73] ggrepel_0.9.8               pillar_1.11.1              
##  [75] stringr_1.6.0               yulab.utils_0.2.5          
##  [77] splines_4.6.1               dplyr_1.2.1                
##  [79] tweenr_2.0.3                treeio_1.37.1              
##  [81] lattice_0.23-1              survival_3.8-12            
##  [83] tidyselect_1.2.1            fontLiberation_0.1.0       
##  [85] knitr_1.52                  RTMB_2.0                   
##  [87] fontBitstreamVera_0.1.1     reformulas_0.4.4           
##  [89] gridExtra_2.3.1             bookdown_0.48              
##  [91] IRanges_2.47.5              Seqinfo_1.3.2              
##  [93] SummarizedExperiment_1.43.0 stats4_4.6.1               
##  [95] xfun_0.62                   Biobase_2.73.2             
##  [97] matrixStats_1.5.0           stringi_1.8.9              
##  [99] lazyeval_0.2.3              ggfun_0.2.2                
## [101] yaml_2.3.12                 boot_1.3-32                
## [103] evaluate_1.0.5              codetools_0.2-20           
## [105] gdtools_0.5.2               tibble_3.3.1               
## [107] BiocManager_1.30.27         ggplotify_0.1.4            
## [109] cli_3.6.6                   FMStable_0.1-4             
## [111] xtable_1.8-8                systemfonts_1.3.2          
## [113] Rdpack_2.6.6                jquerylib_0.1.4            
## [115] dichromat_2.0-1             Rcpp_1.1.2                 
## [117] coda_0.19-4.1               parallel_4.6.1             
## [119] ggplot2_4.0.3               lme4_2.1-0                 
## [121] ggthemes_7.0.0              viridisLite_0.4.3          
## [123] mvtnorm_1.4-2               tidytree_0.4.8             
## [125] ggiraph_0.9.6               lmerTest_3.2-1             
## [127] scales_1.4.0                purrr_1.2.2                
## [129] rlang_1.3.0                 multcomp_1.4-32