# Additional files

Supplementary information accompanying the manuscript:

> Kopp K, Lownds S, Harrison B, Kuhl H, Moittie S. From Field to Genome:
> Non-Lethal, On-Site Nanopore-Only Assembly of the Endangered Grenada Frog
> (*Pristimantis euphronides*). *BMC Genomics*. In submission.

The online version contains supplementary material available at
https://doi.org/10.1186/[DOI to be assigned by BMC].

## Contents

**Additional file 1.** Table. Nomenclatural history, evidentiary basis,
molecular markers, and taxonomic assignments for *Pristimantis euphronides*.

**Additional file 2.** Supplementary materials and methods. Detailed
wet-laboratory and bioinformatic methods including sample collection,
non-lethal sampling protocol, DNA extraction, ONT library preparation and
sequencing, Dorado basecalling, Kraken2 taxonomic screening, de novo assembly
with two independent workflows (Flye and Wtdbg), assembly quality assessment,
reference-guided scaffolding with RagTag, HANNO annotation, sex chromosome
analysis, and 19-gene phylogenomic reconstruction. Includes Tables SM1
(per-library read statistics) and SM2 (sequencing and assembly metadata).

**Additional file 3.** Supplementary results. Extended results for the
*P. euphronides* genome assembly, scaffolding, repeat and gene annotation,
candidate sex-chromosome-linked regions, and phylogenomic analyses. Contains
36 supplementary tables (SR1–SR36) and 31 supplementary figures (SR1–SR31)
covering read-quality metrics, pre- and post-scaffolding assembly statistics,
inter-workflow concordance, Mash-based reference selection, per-scaffold
coverage, repeat landscapes, gene model statistics, Z-candidate region
characterization, tier 1a gametolog identification, W-chromosome candidates,
and supermatrix and single-gene phylogenies.

**Additional file 4.** Supplementary materials and methods — haplotypes.
Methods for the haplotype-resolved diploid assembly cross-check, including
HapDup phasing of the Medaka-polished Flye assembly, PEPPER variant calling,
Margin phasing, Flye haplotype-aware polishing, independent reference-guided
scaffolding of both haplotypes, and inter-haplotype concordance assessment.

**Additional file 5.** Supplementary results — haplotypes. Results of the
haplotype-resolved diploid assembly, including Tables HR1 and HR2 reporting
phasing statistics, per-haplotype assembly metrics, scaffolding outcomes, and
inter-haplotype alignment statistics derived from the deposited D-Genies PAF
files.

**Additional file 6.** Table. Pairwise genomic distances between
*Pristimantis euphronides* and 21 Hyloidea reference genome assemblies.

**Additional file 7.** Figure. Individual scaffold coverage profiles for the
13 longest scaffolds of the *Pristimantis euphronides* genome assembly.

**Additional file 8.** Figure. Scaffolded *Pristimantis euphronides* genome
aligned to *Eleutherodactylus coqui* by chromosome.

**Additional file 9.** Table. Alignment summary for small scaffolds
(scaffolds 14–31) of *Pristimantis euphronides*.

**Additional file 10.** Table. Availability of 18 nuclear gene orthologs
across 20 Hyloidea families.

**Additional file 11.** Phylogenetic analysis using nuclear gene orthologs —
single-gene trees. Twelve maximum-likelihood single-gene phylogenies
(Figures 1–12) of *P. euphronides* and other Hyloidea species inferred from
the nuclear gene orthologs BDNF, BMP2, CXCR4, MC1R, NTF3, POMC, RAG2, RHO,
SIAH1, SLC8A3, TNS3, and TYR, with *Rana temporaria* as outgroup.

**Additional file 12.** Survey of anuran genome assemblies in NCBI Genome.
Comparative survey of all 214 publicly available anuran genome assemblies
(148 species) retrieved from NCBI Genome on 18 April 2026, classified by
sequencing technology, assembly level, taxonomic family, and BioSample tissue
source. Includes Methods, Results, and five Tables (1–5) summarizing the
destructive vs. non-destructive sampling landscape, ONT-only assemblies, and
Terrarana coverage.

## Directory structure

```
Additional_files/
├── Additional_file_1.odt
├── Additional_file_2.odt
├── Additional_file_3.odt
├── Additional_file_4.odt
├── Additional_file_5.odt
├── Additional_file_6.odt
├── Additional_file_7.odt
├── Additional_file_8.odt
├── Additional_file_9.odt
├── Additional_file_10.odt
├── Additional_file_11.odt
├── Additional_file_12.odt
└── Additional_files_README.md
```

## Cross-section dependencies

Tables and figures in the Additional files reference data and results produced
across the analysis directories of this Zenodo deposit. Each Additional file
is self-contained and can be read independently of the main manuscript.

## Data availability

All supplementary data for this study are archived at Zenodo
(doi: [10.5281/zenodo.15298547](https://doi.org/10.5281/zenodo.15298547)).
Scripts are available at GitHub
(https://github.com/koppk/Grenada-Frog-144).

## Author

Kopp K, Pristimantis euphronides genome project
