bio-blast-searches

What this skill does

## Version Compatibility

Reference examples tested with: BioPython 1.83+, NCBI BLAST+ 2.15+

Before using code patterns, verify installed versions match. If versions differ:
- Python: `pip show biopython` then `help(Bio.Blast.NCBIWWW.qblast)` to check signatures
- CLI: `blastn -version` then `blastn -help`

If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying.

# BLAST Searches (Remote)

**"Find similar sequences in NCBI's database"** -> Submit a query to NCBI's remote BLAST servers; receive a Request ID (RID); poll for completion; parse the XML hit table. Best for one-off identification of a few sequences. For >50 sequences, switch to `local-blast` or DIAMOND/MMseqs2 in `remote-homology`.

The two most consequential decisions: **which program** (defines query+target molecule types and word-size defaults) and **which database** (defines the search space and therefore E-value baselines). The third most important: do NOT misuse `max_target_seqs` -- it is an early-termination heuristic, not a "give me the top N hits" filter (Shah et al. 2019).

- Python: `NCBIWWW.qblast(program, db, sequence)` + `NCBIXML.read(handle)` (BioPython)
- CLI: `blastn -remote -db nt -query seq.fa -out hits.xml -outfmt 5` (BLAST+)
- Web: https://blast.ncbi.nlm.nih.gov/Blast.cgi (RID lookup)

## Required Setup

```python
from Bio.Blast import NCBIWWW, NCBIXML
from Bio import SeqIO
```

No API key needed for remote BLAST itself, but NCBI's general rate-limit ethic still applies -- one search at a time, polite waiting, no parallelism.

## Program decision (query vs database molecule)

| Program | Query | Target | Word size default | Use case |
|---|---|---|---|---|
| `blastn` | DNA | DNA | 11 | General DNA similarity |
| `megablast` | DNA | DNA | 28 | High-identity DNA (>=95%) -- PCR primer hits, contamination |
| `dc-megablast` | DNA | DNA | 11 (discontiguous) | Cross-species mRNA (sensitive, gapped) |
| `blastp` | Protein | Protein | 3 (6 also valid) | General protein homology |
| `blastx` | DNA | Protein | 3 | Translated DNA query vs protein DB; ORF discovery |
| `tblastn` | Protein | DNA | 3 | Protein query vs translated DB; find unannotated CDS |
| `tblastx` | DNA | DNA | 3 (both translated) | Most expensive; deep cross-species coding similarity |
| `psiblast` | Protein | Protein | 3 | Iterative PSSM-based remote homology -- see `remote-homology` |

**The misuse to avoid:** using default `blastn` (word=11) for cross-species DNA where `dc-megablast` is the right tool. Or using `megablast` (word=28) for cross-species homology where it will miss every divergent hit. The most-misused BLAST parameter according to literature.

## Database decision (search space)

| Database (`db=`) | Content | Size (2026 approx) | Stable for reproducibility? |
|---|---|---|---|
| `nt` | Non-redundant nucleotide (all GenBank+EMBL+DDBJ) | ~250 GB | NO -- changes daily |
| `nr` | Non-redundant protein | ~300 GB | NO -- changes daily |
| `refseq_select` | One curated rep per species (RNA + protein) | small | YES -- versioned releases |
| `refseq_rna` | RefSeq mRNA | ~10 GB | YES |
| `refseq_protein` | RefSeq protein | small | YES |
| `swissprot` | UniProt Swiss-Prot (reviewed) | small | YES -- monthly releases |
| `pdb` | Protein structures | small | YES |
| `refseq_genomic` | RefSeq genomic | huge | YES |
| `env_nr` / `env_nt` | Environmental (metagenomic) | huge | YES |

**For publication reproducibility, never search `nt` or `nr`** without recording the snapshot date and ideally archiving a frozen copy. Default to `refseq_select` for any cross-species homology question; switch to `nt`/`nr` only when curated coverage is insufficient.

## E-value interpretation (Karlin-Altschul)

E-value = K * m * n * exp(-lambda * S), where m = effective query length, n = effective database size, lambda and K are scoring-matrix-dependent constants (Karlin & Altschul 1990 PNAS 87:2264).

| E-value | Bit-score (BLOSUM62, protein) | Interpretation |
|---|---|---|
| < 1e-50 | > 200 | Strong; almost certainly homologous |
| 1e-50 to 1e-10 | 100-200 | Significant; likely homolog |
| 1e-10 to 1e-3 | 50-100 | Marginal; check identity + coverage |
| 0.01 to 10 | 30-50 | Possible remote homolog; needs profile method |
| > 10 | < 30 | Random; not meaningful |

**Key implication of E = K * m * n * exp(-lambda * S):** the same alignment against a 100x larger database has a 100x larger E-value. Cross-database E-value comparison is meaningless. Bit-score is database-size normalized and is the right cross-database metric.

For protein remote homology where E is marginal (10^-3 to 10^-1), reach for profile methods: PSI-BLAST, jackhmmer, HHblits, or Foldseek -- see `remote-homology` skill.

## Composition-Based Statistics (CBS)

Compositional bias inflates significance for low-complexity proteins. The CBS modes (Yu et al. 2006 *Nucleic Acids Res* 34:5966):

| `composition_based_statistics` | Mode | Use when |
|---|---|---|
| 0 | Off | Almost never |
| 1 | F&S 2002 score adjustment | Legacy compatibility |
| 2 | Yu&Altschul 2005 conditional score adjustment | **Default since BLAST+ 2.2.17** -- correct for most cases |
| 3 | Universal statistics | Short queries (< 30 aa) where mode 2 over-corrects |

For protein queries under 30 aa, switch to CBS=3. For protein with known compositional bias (e.g. coiled-coil regions, signal peptides), CBS=2 is appropriate but consider hard-masking with SEG.

## The `max_target_seqs` trap

**The misuse**: `max_target_seqs=10` is interpreted as "return the 10 most significant hits". It is not. The flag is an **early termination** parameter that affects which hits the search ever considers, not which it ultimately reports (Shah N, Nute MG, Warnow T, Pop M. (2019) Misunderstood parameter of NCBI BLAST impacts the correctness of bioinformatics workflows. *Bioinformatics* 35:1613-1614).

**Consequences:**
- Setting `max_target_seqs=10` can return entirely different hits than `max_target_seqs=500` then filtering to top 10 by E-value.
- The "top 10" by E-value as reported may not be the actual top 10.

**Correct pattern:** set `hitlist_size` (Bio.Blast parameter name) large (1000+), then post-filter to the top N by E-value or bit-score in Python.

## Word size, gap costs, and matrix

| Search | Word size | Matrix (protein) | Gap (open, extend) |
|---|---|---|---|
| megablast (high identity DNA) | 28 | n/a | 0, 0 (linear) |
| blastn (sensitive DNA) | 11 | n/a | 5, 2 |
| blastp default | 3 | BLOSUM62 | 11, 1 |
| blastp distant | 2 | BLOSUM45 | 14, 2 |
| Short peptides (<30 aa) | 2 | PAM30 or BLOSUM45 | 9, 1 |

For very short query proteins (e.g. proteomics-identified peptides), BLOSUM45 + word=2 + PAM30 substitution matrix is more sensitive than the default. Use `matrix='PAM30'` for searches against `swissprot`.

## RID lifecycle

| Phase | Server state | Client action |
|---|---|---|
| Submit | RID created, queued | NCBIWWW.qblast() returns handle |
| Running | Queue + compute | Poll status |
| Done | RID + results retained | Fetch XML |
| Expired | RID purged | 24-36h after completion |

`NCBIWWW.qblast()` handles polling internally with a fixed retry interval. For long-running searches (>5 min) or batches, submit and capture the RID, then poll independently to avoid blocking. The RID is visible at `https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=...` for 24-36 hours.

## Code patterns

### Standard remote BLASTN with reproducible parameters

**Goal:** Run BLASTN with explicit, paper-quality parameters.

**Approach:** Specify program, database (refseq_select for stability), word size, expect, and a large hitlist_size to dodge the max_target_seqs trap.

**Reference (BioPython 1.83+):**
```python
from Bio.Blast import NCBIWWW, NCBIXML

handle = NCBIWWW.qblast(
    program='blastn',
    database='refseq_select_rna',
    sequence=query_seq,
    expect=1e-10,
    word_size=11,
    hitlist_size=500,  # la