| name | bio-small-rna-seq-target-prediction |
| description | Predicts and prioritizes miRNA target genes with seed-based tools (miRanda, TargetScan, miRDB) and experimentally validated databases (miRTarBase, multiMiR). Use when deciding that a predicted target is a hypothesis not a finding; ranking by the right score (weighted context++, mirSVR, miRDB); raising confidence by intersecting predictions with inversely-correlated mRNA DE; weighing validated (CLIP/reporter) over predicted evidence; or avoiding the circular enrichment of unfiltered target lists. |
| tool_type | mixed |
| primary_tool | miRanda |
Version Compatibility
Reference examples tested with: miRanda 3.3a+, BioPython 1.83+, pandas 2.2+, gseapy 1.1+
Before using code patterns, verify installed versions match. If versions differ:
- CLI:
<tool> --version then <tool> --help to confirm flags
- Python:
pip show <package> then help(module.function) to check signatures
If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying.
miRNA Target Prediction
"Predict target genes for my miRNAs" -> Generate candidate mRNA targets by seed complementarity and thermodynamics, then raise confidence with conservation, validated databases, and matched expression.
- CLI:
miranda miRNA.fa UTR.fa -sc 140 -en -20 -strict for de novo prediction
- Python: query TargetScan/miRDB downloads and miRTarBase for validated interactions
The governing principle: a predicted target is a hypothesis, not a finding
Seed-based prediction has a false-positive rate near 50% even for conserved sites (Pinzon 2017), because a 6mer seed match occurs by chance roughly once per 4 kb of sequence, so a multi-kb 3' UTR carries many spurious matches. It also has a RECALL problem in the opposite direction: AGO-CLIP and CLASH show that roughly 60% of real interactions are noncanonical, and the bulged, seedless, and 3'-compensatory sites among them are missed entirely by seed-only tools (3'-supplementary sites keep a canonical seed and are still found), so a clean seed list is incomplete, not just imprecise (Helwak 2013). Worse, a single miRNA represses most of its real targets only modestly - typically less than two-fold at the protein level (Baek 2008; Selbach 2008) - so miRNAs are rheostats, not switches, and large single-target claims should be distrusted. The decisive move is therefore not running more predictors (five seed-based tools agreeing is pseudo-replication, not independent evidence) but climbing an evidence ladder and, above all, intersecting predictions with INVERSELY-correlated differentially expressed mRNA or protein from the SAME samples. Prediction proposes; expression disposes.
| Evidence tier (low to high) | What it means |
|---|
| seed match alone | weakest; common by chance |
| + conservation (TargetScan PCT) | precision up, recall down (misses species-specific targets) |
| + multiple independent tools / ML (miRDB) | modestly higher precision |
| + AGO-CLIP footprint | the miRNA's complex bound there (but CLIP is cell-type/state-specific - a peak from another tissue is weak evidence) |
| + CLASH/CLEAR-CLIP chimera | direct miRNA-target duplex |
| + anti-correlated matched miRNA/mRNA(protein) DE | functional in YOUR system |
| + reporter / seed-mutation rescue (miRTarBase "strong") | causal, gold standard |
Decision: which predictor, and how it scores
| Tool | Scoring philosophy | Use for | Caveat |
|---|
| TargetScan (context++) | conservation + 14-feature regression of repression | conserved-site prioritization | v7 = context++; v8 = a different Kd/biochemical model |
| miRanda + mirSVR | thermodynamic alignment + expression-trained regression | non-conserved / non-canonical sites | permissive; tune thresholds |
| miRDB / MirTarget | ML (SVM) on CLIP + overexpression data | data-driven ranking (score 0-100) | score >= 80 is the conventional high-confidence cut |
| RNAhybrid | pure MFE hybridization, no seed constraint | exploratory, no-seed sites | most false positives without filters |
| miRTarBase / TarBase (ENCORI) | experimentally validated interactions | the gold tier; anchor claims here | "less strong" CLIP/NGS entries are not individually validated |
| multiMiR | unifies predicted + validated sources | one-call aggregation | inherits each source DB's errors |
De novo prediction with miRanda
Goal: Predict miRNA-mRNA target sites by complementarity and duplex energy.
Approach: Align miRNA sequences against 3' UTRs with a minimum score and a maximum (negative) energy, requiring strict seed pairing.
miranda miRNA.fa UTRs.fa -sc 140 -en -20 -strict -out predictions.txt
Parse miRanda output
Goal: Extract interaction records into a DataFrame.
Approach: Read the lines miRanda prefixes with '>' (per-hit summary) and pull miRNA, target, score, and energy.
import pandas as pd
def parse_miranda(output_file):
rows = []
with open(output_file) as f:
for line in f:
if line.startswith('>') and not line.startswith('>>'):
p = line.strip().split('\t')
if len(p) >= 5:
rows.append({'mirna': p[0].lstrip('>'), 'target': p[1],
'score': float(p[2]), 'energy': float(p[3])})
return pd.DataFrame(rows)
TargetScan context-scores lookup
Goal: Retrieve conserved-site predictions and rank a miRNA's targets.
Approach: Read the downloadable per-site context-scores file and rank by the weighted context++ score (more negative = stronger predicted repression across the whole UTR).
import pandas as pd
def query_targetscan(mirbase_id, ts_file='Predicted_Targets_Context_Scores.default_predictions.txt'):
df = pd.read_csv(ts_file, sep='\t')
hits = df[df['Mirbase ID'] == mirbase_id]
return hits.sort_values('weighted context++ score')
miRDB (machine-learning) lookup
Goal: Retrieve ML-based target predictions above the conventional confidence cut.
Approach: Read the miRDB prediction download and keep targets with score >= 80.
def query_mirdb(mirna_id, mirdb_file='miRDB_v6.0_prediction_result.txt'):
df = pd.read_csv(mirdb_file, sep='\t', header=None, names=['mirna', 'refseq', 'score'])
hits = df[df['mirna'] == mirna_id]
return hits[hits['score'] >= 80].sort_values('score', ascending=False)
Validated targets and unified lookup
Goal: Anchor target claims in experimental evidence rather than prediction.
Approach: Query miRTarBase for validated interactions and weight by evidence type; use multiMiR (R) to unify predicted and validated sources in one call.
def get_validated_targets(mirna, mirtarbase_file='miRTarBase_MTI.xlsx'):
df = pd.read_excel(mirtarbase_file)
hits = df[df['miRNA'] == mirna]
return hits[['Target Gene', 'Experiments', 'Support Type']]
The confidence move: intersect with anti-correlated mRNA DE
Goal: Keep only targets that behave functionally in the actual experiment.
Approach: Intersect predicted (or CLIP-supported) targets of UP miRNAs with DOWN mRNAs from matched samples; note the blind spot that translation-only targets may not move at the mRNA level.
def functional_targets(predicted_targets, mrna_de, mirna_direction):
want_down = mirna_direction == 'up'
moved = mrna_de[(mrna_de['log2FC'] < 0) == want_down].index
return [g for g in predicted_targets if g in set(moved)]
Seed match analysis and site types
Goal: Locate seed matches in a UTR and classify site strength.
Approach: The seed is miRNA positions 2-7; a site is the reverse complement of the seed in the 3' UTR. Canonical sites by decreasing efficacy: 8mer > 7mer-m8 > 7mer-A1 > 6mer.
from Bio.Seq import Seq
def find_seed_matches(mirna_seq, utr_seq):
seed = str(Seq(mirna_seq)[1:8])
site = str(Seq(seed).reverse_complement())
matches, start = [], 0
while True:
pos = utr_seq.find(site, start)
if pos == -1:
break
matches.append(pos)
start = pos + 1
return matches
Common Errors
| Symptom | Cause | Fix |
|---|
KeyError: 'miRNA family' on TargetScan file | Wrong column name for the context-scores file | The miRNA column is Mirbase ID; rank by weighted context++ score |
| Hundreds of "targets", almost none real | Treating seed prediction as truth | Intersect with anti-correlated mRNA DE; anchor in miRTarBase strong evidence |
| Every miRNA "regulates cancer pathways" | Enrichment on an unfiltered predicted target list (circular) | Build the list from validated/CLIP or expression-filtered targets before enrichment |
| Five tools "agree" so a target is trusted | All five use the seed (pseudo-replication) | Require an orthogonal evidence tier (CLIP/validated/expression), not more seed tools |
| A strong single-target claim | miRNAs repress most targets < 2-fold | Treat large single-target effects skeptically; demand validation |
| ceRNA/sponge mechanism asserted | Stoichiometry usually too low to matter (Denzler) | Require absolute abundance (miRNA copies vs added sites) before accepting it |
Related Skills
- differential-mirna - Source of the DE miRNAs to predict targets for
- pathway-analysis/go-enrichment - Enrich a target list (only after evidence-filtering)
- database-access/entrez-fetch - Fetch UTR/gene sequences and identifiers
- clip-seq/ago-clip-mirna-targets - AGO-CLIP / CLASH direct target evidence
References
- Agarwal V, Bell GW, Nam JW, Bartel DP. 2015. Predicting effective microRNA target sites in mammalian mRNAs. eLife 4:e05005. doi:10.7554/eLife.05005
- Betel D, Koppal A, Agius P, Sander C, Leslie C. 2010. Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites. Genome Biol 11:R90. doi:10.1186/gb-2010-11-8-r90
- Chen Y, Wang X. 2020. miRDB: an online database for prediction of functional microRNA targets. Nucleic Acids Res 48:D127-D131. doi:10.1093/nar/gkz757
- Huang HY, Lin YC, Cui S, et al. 2022. miRTarBase update 2022: an informative resource for experimentally validated miRNA-target interactions. Nucleic Acids Res 50:D222-D230. doi:10.1093/nar/gkab1079
- Ru Y, Kechris KJ, Tabakoff B, et al. 2014. The multiMiR R package and database: integration of microRNA-target interactions. Nucleic Acids Res 42:e133. doi:10.1093/nar/gku631
- Pinzón N, Li B, Martinez L, et al. 2017. microRNA target prediction programs predict many false positives. Genome Res 27:234-245. doi:10.1101/gr.205146.116
- Baek D, Villén J, Shin C, et al. 2008. The impact of microRNAs on protein output. Nature 455:64-71. doi:10.1038/nature07242
- Selbach M, Schwanhäusser B, Thierfelder N, et al. 2008. Widespread changes in protein synthesis induced by microRNAs. Nature 455:58-63. doi:10.1038/nature07228
- Helwak A, Kudla G, Dudnakova T, Tollervey D. 2013. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153:654-665. doi:10.1016/j.cell.2013.03.043
- Denzler R, Agarwal V, Stefano J, Bartel DP, Stoffel M. 2014. Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. Mol Cell 54:766-776. doi:10.1016/j.molcel.2014.03.045
