Bioinformatics Summary

Fundamentals and Scope of Bioinformatics

  • Definition: An interdisciplinary field merging biology with computer science, information technology, mathematics, and statistics to store, manage, analyze, and interpret complex biological data.

  • Core Components:

    • Developing computational algorithms, software tools, and database storage architectures.

    • Applying computational tools to analyze biological systems and generate biological insights.

  • Sequencing Cost Reduction: Advancements in high-throughput sequencing drastically dropped human whole-genome sequencing costs from roughly $2.7 billion\$2.7\text{ billion} in 2003 to $300,000\$300{,}000 in 2006, and down to $1,000\$1{,}000 in 2016.

Biological Databases

  • Primary Databases: Store original, directly submitted experimental sequence data:

    • GenBank: National Center for Biotechnology Information (NCBI).

    • ENA (European Nucleotide Archive): EMBL-EBI.

    • DDBJ: DNA Data Bank of Japan.

    • UniProt: Universal Protein Resource, housing protein sequence and functional annotation.

  • Secondary Databases: Curate and compile derived information, patterns, and conserved motifs from primary databases (e.g., Pfam, InterPro).

  • RefSeq (NCBI Reference Sequence Database): Provides a curated, non-redundant, centralized collection of reference sequences covering genomic DNA, transcripts, and proteins across diverse organisms.

  • Accession Numbers: Unique alphanumeric identifiers (e.g., U03187.1, X25684.1) assigned to specific sequence records to ensure direct access across platforms.

Sequence Formats and Alignment Notation

  • FASTA Format: Standard text format beginning with a single-line description initiated by a > character, followed by raw nucleotide or amino acid sequence data on subsequent lines without internal spaces.

FASTA sequence format

  • Multiple Sequence Alignment (MSA) Formats: Standardized structures used by tools such as CLUSTAL (e.g., ClustalW, Clustal Omega), NEXUS, and PHYLIP.

  • CLUSTAL Alignment Output Symbols:

    • * (asterisk): Single, fully conserved residue across all aligned sequences.

    • : (colon): Strongly conserved substitutions with similar biochemical properties (PAM 250 score>0.5\text{PAM 250 score} > 0.5).

    • . (period): Weakly conserved substitutions with weakly similar properties (PAM 250 score≤0.5\text{PAM 250 score} \le 0.5).

Sequence Alignment Tools

  • BLAST (Basic Local Alignment Search Tool): Identifies local regions of statistical similarity between a query sequence and database targets:

    • blastn: Compares nucleotide query against nucleotide database.

    • blastp: Compares protein query against protein database.

    • blastx: Translates nucleotide query in six frames and compares against protein database.

    • tblastn: Compares protein query against six-frame translated nucleotide database.

  • BLAT (BLAST-Like Alignment Tool):

    • Developed by Jim Kent at UCSC for human genome assembly and cross-species comparisons.

    • Operates ∼500×\sim 500\times faster than BLAST for mRNA/DNA alignments and ∼50×\sim 50\times faster for protein/protein alignments.

    • Optimized for rapid mapping of mRNA/EST sequences to genomes, identifying exon-intron boundaries, and detecting gene family members.

Peptide Annotation and Protein Feature Prediction

  • Conceptual Translation & Reading Frames:

    • Double-stranded DNA contains six potential reading frames (33 forward and 33 reverse).

    • The biologically meaningful frame that translates into an unbroken peptide sequence without premature stop codons is designated the Open Reading Frame (ORF).

Three reading frames of a nucleic acid sequence

  • Gene Ontology (GO): Standardized, species-independent classification of gene product attributes across three domains:

    • Cellular Component: Subcellular localization or macromolecular complex (e.g., nucleus, membrane).

    • Molecular Function: Biochemical activity at the molecular level (e.g., catalytic activity, binding).

    • Biological Process: Coordinated series of molecular events (e.g., heart contraction, signal transduction).

  • Signal Peptides: Conserved N-terminal targeting peptides (∼30\sim 30 amino acids) directing protein sorting; characterized by a positively charged N-terminus, a central hydrophobic core, an uncharged polar C-terminus, and a conserved cleavage site.

  • Transmembrane (TM) Domains: Extended stretches of hydrophobic amino acids spanning lipid bilayers, identified via computational hydrophobicity algorithms.

Principles of Phylogenetics

  • Phylogenetic Trees: Branching diagrams illustrating evolutionary histories and divergence among species, genes, or populations.

Phylogenetic tree terminology

  • Tree Anatomy:

    • Root: Ancestral node representing the most recent common ancestor of all taxa in the tree.

    • Internal Nodes: Divergence points representing inferred ancestral lineages.

    • Branches (Lineages): Depict ancestral-descendant relationships over evolutionary time.

    • Terminal Nodes (Taxa): Extant or analyzed operational taxonomic units (genes, populations, species).

  • Molecular vs. Morphological Data: Molecular sequence data (DNA, RNA, proteins) provides unambiguous, highly specific, and reliable phylogenetic reconstructions compared to morphological traits, which can be confounded by convergent evolution.

  • Applications: Organism classification, forensic identification, tracing pathogen transmission and emergence, conservation planning, and phylogenetic software development.