design_xenium_probes
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Design Xenium custom-panel padlock targets on the subject transcript to 10x Genomics' published rules (CG000683): two 20 nt arms whose primer3 melting temperatures fall in their windows, a ligation-junction dinucleotide the Xenium ligase accepts, no run of five G in the probe, at least four mismatches across both arms to every other transcript in a BLAST search, and probes at least 10 bp apart. Without BLAST results it returns the targets to search and says the off-target check has not been run.
Input Schema
| Name | Required | Description | Default |
|---|---|---|---|
| _files | No | The project: record references shaped like _subject. Their records become library documents a tool names in sequences/parts/document; a trace or alignment is found by file name. | |
| spacing | No | fewest bases between two kept targets. In bp. | |
| tm_rule | No | which melting temperatures must fall in their windows. 10x: the full length between 68 and 82 °C or each arm between 50 and 70 °C. | either |
| _subject | No | The record to act on: exactly one of sequence, accession (+source), sample (an id from list_samples) or file ({name, text} or {name, base64}); record_index picks one record of several. | |
| blast_csv | No | blastn -outfmt 10 output for the targets FASTA this tool returns, searched against the transcriptome (not the genome) with -word_size 7 -strand plus. Without it no target is checked for off-target homology. | |
| junctions | No | ligation-junction dinucleotides to accept, from 10x's empirical ligase preferences; 10x strongly recommends avoiding the nonpreferred four. | preferred and neutral |
| max_g_run | No | longest run of G the probe may carry; 10x: avoid five or more. Advanced. | |
| arm_length | No | length of each arm; the target is twice this. 10x's arms are 20 nt, shorter only for a short target whose Tm stays in range. In nt. Advanced. | |
| max_arm_tm | No | highest Tm of each arm (inclusive). In °C. | |
| min_arm_tm | No | lowest Tm of each arm (inclusive). In °C. | |
| prefer_cds | No | rank targets inside the subject's CDS feature before the untranslated regions, as 10x advises where possible. | |
| max_full_tm | No | highest Tm of the whole target (inclusive). In °C. | |
| min_full_tm | No | lowest Tm of the whole target (inclusive). In °C. | |
| min_mismatches | No | fewest mismatches across both arms an off-target hit must carry for the target to be kept; gaps and unaligned ends count. Only applies when blast_csv is set. | |
| gene_accessions | No | comma-separated accessions (no version) that are this gene, its isoforms included: hits to them are not off-targets. Empty: the subject's name. Only applies when blast_csv is set. | |
| probes_per_gene | No | targets to keep; 10x's predesigned panels use eight per gene. |
Output Schema
| Name | Required | Description | Default |
|---|---|---|---|
| probes | Yes | the kept targets, each with its 0-based start, target (mRNA sense), probe (its reverse complement), arm and full-length Tm, junction and its class, and the fewest off-target mismatches. Null until `blast_csv` is given | |
| stages | Yes | what each rule took in and kept, in order, with the reason a skipped check did not run | |
| probeCount | Yes | how many targets were kept. Null until `blast_csv` is given | |
| explanation | Yes | the derivation chain: conclusion, interpretation and machinery, generated from this run | |
| targetsFasta | Yes | the targets that pass the Tm, junction and G-run rules as FASTA, named xenium_<1-based start>, for blastn |