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7 | 7 |
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8 | 8 | ## Pipeline overview (ordered) |
9 | 9 | 1. **Read FASTA record(s).** |
10 | | - - `designProbes` uses only the first FASTA record. |
11 | | - - `designProbesBatch` processes every record. |
12 | | - - A FASTA header can override the channel using `channel=...`. |
13 | | - - Genome masking is enabled by default and requires a registered species |
14 | | - (use `fetchMouseIndex` or `buildGenomeIndex`) or an explicit `--index`. |
| 10 | + |
| 11 | + - `designProbes` uses only the first FASTA record. |
| 12 | + - `designProbesBatch` processes every record. |
| 13 | + - A FASTA header can override the channel using `channel=...`. |
| 14 | + - Genome masking is enabled by default and requires a registered species |
| 15 | + (use `fetchMouseIndex` or `buildGenomeIndex`) or an explicit `--index`. |
15 | 16 | 2. **Optional repeat masking (disabled by default).** |
16 | | - - If enabled, the input sequence is masked using RepeatMasker and masked bases |
17 | | - are converted to `N`. Tiles containing masked bases are discarded later. |
| 17 | + |
| 18 | + - If enabled, the input sequence is masked using RepeatMasker and masked bases |
| 19 | + are converted to `N`. Tiles containing masked bases are discarded later. |
18 | 20 | 3. **Tile the target sequence (reverse-complemented).** |
19 | | - - The sequence is scanned with a step size of 1 to generate 52-nt tiles by |
20 | | - default. |
21 | | - - Each tile is **reverse-complemented** so probes are antisense to the target. |
22 | | - - Any tile containing `N` is discarded immediately. |
| 21 | + |
| 22 | + - The sequence is scanned with a step size of 1 to generate 52-nt tiles by |
| 23 | + default. |
| 24 | + - Each tile is **reverse-complemented** so probes are antisense to the target. |
| 25 | + - Any tile containing `N` is discarded immediately. |
23 | 26 | 4. **Filter homopolymer runs (C and G).** |
24 | | - - Tiles are removed if they contain long runs of C's or G's beyond the allowed |
25 | | - thresholds. |
| 27 | + |
| 28 | + - Tiles are removed if they contain long runs of C's or G's beyond the allowed |
| 29 | + thresholds. |
26 | 30 | 5. **Filter hairpins.** |
27 | | - - Each tile is screened with `primer3` for self-hairpin structures. |
28 | | - - Tiles with a predicted hairpin melting temperature >= 45 C are removed. |
| 31 | + |
| 32 | + - Each tile is screened with `primer3` for self-hairpin structures. |
| 33 | + - Tiles with a predicted hairpin melting temperature >= 45 C are removed. |
29 | 34 | 6. **Genome uniqueness filtering (Bowtie2).** |
30 | | - - Remaining tiles are aligned to the reference genome index. |
31 | | - - Tiles with more than the allowed number of hits are removed. |
| 35 | + |
| 36 | + - Remaining tiles are aligned to the reference genome index. |
| 37 | + - Tiles with more than the allowed number of hits are removed. |
32 | 38 | 7. **GC-content filtering.** |
33 | | - - Tiles must fall within the allowed GC% range. |
| 39 | + |
| 40 | + - Tiles must fall within the allowed GC% range. |
34 | 41 | 8. **Gibbs free energy filtering.** |
35 | | - - Tiles with binding free energies outside the allowed range are removed. |
| 42 | + |
| 43 | + - Tiles with binding free energies outside the allowed range are removed. |
36 | 44 | 9. **Split tiles into probe halves.** |
37 | | - - Each tile is split into two 25-mers (for a 52-mer tile), dropping the two |
38 | | - middle bases to create a short gap. |
| 45 | + |
| 46 | + - Each tile is split into two 25-mers (for a 52-mer tile), dropping the two |
| 47 | + middle bases to create a short gap. |
39 | 48 | 10. **Optional dTm filtering.** |
| 49 | + |
40 | 50 | - If enabled, tiles are removed when the temperature difference between |
41 | 51 | the two halves is too large. |
42 | 52 | 11. **Select top N non-overlapping tiles.** |
| 53 | + |
43 | 54 | - Tiles are ranked by how close their Gibbs free energy is to the target. |
44 | 55 | - Overlapping tiles are skipped to keep probes spread out. |
45 | 56 | 12. **Add HCR initiators and spacers.** |
| 57 | + |
46 | 58 | - Channel-specific initiator sequences are appended to the probe halves. |
47 | 59 |
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48 | 60 | ## Default parameters |
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