Large reference inputs for the entropy-guided fish eDNA primer design pipeline

These are the large files referenced by the analysis code at https://github.com/sundew555/fish-edna-universal-primers, which are too large to distribute through git. Place them in data/primer_design/ (or set the UNIPRIMER_DATA environment variable) and every script resolves them automatically through config.py. Everything else the pipeline needs, including both primer-design routes and their small inputs, is in the repository itself. Contents mitogenome_sequences.fasta (69 MB, 4,213 sequences) — the unaligned fish mitogenome set, FASTA headers carrying accession and taxid. This is both the input to the alignment step and the reference for every in silico amplification step. mitogenome_alignment.fasta (68 MB, 4,214 sequences) — MAFFT --keeplength reference-guided alignment of that set, anchored on NC_001606.1 (Cyprinus carpio). The 4,214 records are the 4,213 mitogenomes plus the anchoring reference. primer_screening_results.xlsx (24 MB, 2,699 rows) — route A's candidate pairs that entered in silico screening: the top 200 scoring 20-nt pairs per mitochondrial gene, each annotated with the number of reference species amplified under the repository's matching rules. selected_primer_candidates.xlsx (57 rows) — the curated shortlist assembled from both design routes, with per-candidate amplification counts. Under its internal numbering, candidate Hui7 is the pair reported as Hu and candidate Hui1 is the pair reported as Hui. The two design routes The study used two primer-design routes in parallel and carried the best candidate from each into laboratory testing. Route A: exhaustive enumeration of 20-nt candidates, scored by the entropy of the priming windows; its candidate table is file 3 above; produced Hu (12S). Route B: entropy-thresholded conserved windows followed by Primer3 design at 18-25 nt, with all its inputs and outputs in the repository; produced Hui (16S), 21 + 21 nt. Because route B allows primers longer than 20 nt, Hui does not, and cannot, appear in primer_screening_results.xlsx. Provenance Files 1 and 2 are byte-identical copies of the files used for the analyses in the manuscript; only their filenames were changed, to match the repository. In files 3 and 4, three column headers that were originally in Chinese were renamed to the English names the published scripts expect (amplified_species_count, amplified_species_ids, distinct_amplicon_count). No values were altered. Running the selection logic of 06_select_top_primers.py on primer_screening_results.xlsx reproduces the 150 primer pairs in the repository's data/primer_design/top_primers_per_gene.xlsx exactly. A full README accompanies the files. The underlying mitogenome sequences are public NCBI RefSeq records and carry no additional restrictions.