The Basic Local Alignment Search Tool, commonly known as BLAST, is the standard method in bioinformatics for comparing biological sequences, whether they are made of DNA, RNA, or protein. Researchers use this tool to take a query sequence and search for similar sequences within massive public databases, helping to reveal genetic relationships and potential functions. The results provide a set of metrics that quantify the quality of each match, and among these, the Expectation Value, or E-value, stands out as the most informative statistical measure. Understanding the E-value provides a clear, objective measure of the match’s significance, indicating whether the alignment is a meaningful biological discovery or merely a random occurrence.
BLAST: The Foundation of Sequence Searching
BLAST serves as a sophisticated search engine for molecular data, designed to efficiently find regions of similarity between a user’s input sequence and the hundreds of millions of sequences stored in global repositories. The tool works by rapidly identifying short, highly similar segments, called “words,” between the query and the database sequences. It then extends these words into longer, high-scoring alignments, creating a map of potential genetic or protein relationships.
This capability allows scientists to infer the function of a newly discovered gene by finding its relatives in well-studied organisms. The output presents a table of hits, each associated with metrics like the percentage of identical residues and a raw alignment score. However, the raw score alone does not account for the size of the database, which is where the E-value provides statistical correction.
Defining the Expectation Value (E-value)
The Expectation Value (E-value) is a statistical parameter that quantifies the number of matches with a score equal to or better than the observed alignment score that one would expect to find by chance. This metric models the random background noise of the search space. For example, an E-value of 1.0 means the search is expected to return one random hit of that quality in the database. Conversely, an E-value of 0.01 suggests that only one such random match is expected for every 100 searches performed. A lower E-value corresponds to a match that is less likely to be random noise, suggesting a strong likelihood of true biological or evolutionary relatedness.
Interpreting E-value Results
The practical interpretation of the E-value is guided by a simple principle: the closer the value is to zero, the more confident a researcher can be that the match is meaningful. Highly significant results are often reported using scientific notation, such as 1e-50, which is shorthand for $1 \times 10^{-50}$. E-values in this range are considered evidence of a near-perfect match or a very close evolutionary relationship. Researchers often apply a significance threshold, a cutoff value used to filter out noise and retain only the most reliable hits. Common thresholds are set at $1 \times 10^{-3}$ or $1 \times 10^{-5}$; any alignment with an E-value greater than this limit is usually discarded as statistically insignificant.
Factors That Shape the E-value
The E-value is a dynamic statistic heavily influenced by the context of the search, which is why the same alignment score can yield different E-values depending on the parameters used. One of the primary variables is the size of the sequence database being queried. Searching a larger database increases the overall search space, which in turn increases the probability of finding a high-scoring match purely by chance. Consequently, a larger database will result in a higher (less significant) E-value for a given alignment score, acting as a correction for the increased chance of random hits.
The length of the query sequence also has a substantial impact on the calculated E-value. Shorter query sequences are inherently more likely to align randomly with sequences in the database compared to much longer sequences. Due to this statistical reality, a short sequence that aligns perfectly will receive a higher E-value than a much longer sequence with the same perfect alignment score.