Sequence Properties

Hard rules

1. No fabricated citations. Every cited work must resolve to a verifiable
2. No claim without provenance. Every quantitative or factual claim
3. No silent failure. Every script invocation, API call, or tool use must declare its exit status and what to do on non-zero. A skill that silently swallows errors is a violation.

When to use

• Pre-alignment sanity checks (a sequence with 90% Ns is not real).
• Primer/probe design: Tm, GC, hairpin, dimer.
• Protein characterization: MW, pI, instability, aromaticity, GRAVY.
• Genome composition: GC content by window, by chromosome, by gene.

When NOT to use

• Production primer design → use primer3-py (handles salt, oligo concentration, mismatches).
• Genome-wide GC → use bedtools nuc or computeGCBias (deepTools).
• Protein domain detection → use InterProScan or pyhmmer.

Prerequisites

• biopython>=1.83
• For protein: Bio.SeqUtils.ProtParam
• For DNA/RNA: Bio.SeqUtils (nt utilities)

Core workflow

1. Identify the molecule type (DNA, RNA, protein).
2. Pick the right utility module — ProtParam is for protein, the rest of Bio.SeqUtils for DNA/RNA.
3. Compute the canonical properties for that type.
4. Cite the formula in your methods section.

Code patterns

DNA / RNA: GC, MW, Tm

from Bio.Seq import Seq
from Bio.SeqUtils import gc_fraction
from Bio.SeqUtils.MolecularWeight import MolecularWeight
"
s = Seq("ATGCATGCATGCATGCATGC")
print(f"length: {len(s)}")
print(f"GC: {gc_fraction(s):.3f}")  # 0.5

mw_double = MolecularWeight(s)         # double-stranded MW
print(f"ds MW: {mw_double:.1f} Da")

DNA Tm (Wallace rule for short oligos)

For very short oligos (<14 nt), Tm is roughly 2 * (A+T) + 4 * (G+C):

def tm_wallace(seq: str) -> float:
    s = seq.upper()
    return 2 * (s.count("A") + s.count("T")) + 4 * (s.count("G") + s.count("C"))

DNA Tm (Marmur / nearest-neighbor for longer oligos)

Use primer3-py for production work — see the primer-design reference. The nearest-neighbor model (SantaLucia 1998) is the 2026 standard.

# In production: primer3-py
import primer3
tm = primer3.calc_tm("ATGCATGCATGCATGC")

Protein: MW, pI, instability, aromaticity, GRAVY

from Bio.Seq import Seq
from Bio.SeqUtils.ProtParam import ProteinAnalysis

pa = ProteinAnalysis(str(Seq("MGEKLPVRLNVMGYEEDILKQHKWLRNVQTLKDGIVFVD")))

print(f"length: {len(pa.sequence)}")
print(f"MW: {pa.molecular_weight():.1f} Da")
print(f"pI: {pa.isoelectric_point():.2f}")
print(f"instability_index: {pa.instability_index():.1f}")  # <40 stable
print(f"aromaticity: {pa.aromaticity():.3f}")
print(f"gravy: {pa.gravy():.3f}")  # Grand average of hydropathy

Amino acid percent composition

print(pa.get_amino_acids_percent())
# {'A': 0.07, 'C': 0.02, ...}

Secondary structure fraction (Chou-Fasman)

print(pa.secondary_structure_fraction())  # (helix, turn, sheet)

GC content by sliding window

from Bio.Seq import Seq
from Bio.SeqUtils import gc_fraction

def gc_window(seq: Seq, window: int = 100, step: int = 50):
    s = str(seq)
    out = []
    for i in range(0, len(s) - window + 1, step):
        out.append((i, gc_fraction(Seq(s[i:i+window]))))
    return out

CpG observed/expected

CpG O/E = (CpG count) / (C count × G count / N). Used in vertebrate methylation studies.

def cpg_oe(s: str) -> float:
    s = s.upper()
    c = s.count("C")
    g = s.count("G")
    cg = s.count("CG")
    n = c + g
    if c == 0 or g == 0 or n == 0:
        return 0.0
    return cg * n / (c * g)

DNA / RNA / protein ambiguity

gc_fraction excludes N automatically. If you need a denominator that includes Ns, normalize manually:

def gc_with_n(s: str) -> float:
    s = s.upper()
    denom = sum(1 for b in s if b in "ACGTNU")
    gc = sum(1 for b in s if b in "GC")
    return gc / denom if denom else 0.0

Common pitfalls

• Bio.SeqUtils.GC vs gc_fraction. In 1.80+, the canonical function is gc_fraction(seq). Older code uses GC(seq). The new function returns a fraction; old returned a percentage.
• Wallace Tm is wrong for oligos > 14 nt. Use nearest-neighbor (SantaLucia 1998) for production.
• Tm calculation is salt-, oligo-concentration-, and Mg2+-dependent. A "Tm" without conditions is meaningless. primer3-py handles this.
• Instability index is for in vitro stability of a purified protein, not cellular half-life. Don't conflate.
• GRAVY negative = hydrophilic, positive = hydrophobic. The sign convention trips people up.

Validation

• Length: len(seq) == len(str(seq)).
• GC: 0 ≤ GC ≤ 1.
• pI: amino acid distribution implies a pI; verify with the expected range for the protein class (e.g., basic proteins have pI > 7).
• MW: protein MW = sum of residue masses + 18 (water). ProteinAnalysis accounts for this.

Open alternatives

References

• Biopython ProtParam: https://biopython.org/docs/latest/api/Bio.SeqUtils.ProtParam.html
• SantaLucia 1998 nearest-neighbor Tm: https://doi.org/10.1073/pnas.95.4.1460
• ExPASy ProtParam: https://web.expasy.org/protparam/
• Companion: ors-bioinformatics-sequence-codon-usage, ors-bioinformatics-sequence-reverse-complement.

Changelog

• 1.0.0 (2026-06-10): Initial adaptation by Pradyumna Jayaram from bio-sequence-properties (bioSkills-main/sequence-manipulation/sequence-properties).

Cross-references

Other skills in this category:

• batch-processing
• bowtie2-alignment
• bwa-alignment
• bwa-mem2-alignment
• codon-usage
• compressed-sequence-files
• fastq-quality-scores
• filter-sequences
• format-conversion
• hisat2-alignment
• motif-search
• paired-end-fastq
• pysam-genomics
• read-write-sequences
• reverse-complement
• sam-bam-basics
• samtools-bam-processing
• seq-objects
• sequence-slicing
• sequence-statistics
• star-alignment
• transcription-translation