WHAT IS AN E-VALUE?
A BLASTn search returns hits, sequences that produce “significant” alignments to the
query sequence. The significance of a hit is measured by its E-value, or expect value.
Each alignment has a bit score (S), which is a measure of similarity between the hit and
the query; it is given in the column next to the E-value. The E-value of a hit is the number
of alignments with bit score  S that you expect to find by chance (i.e., with no
evolutionary explanation). Biologically significant hits will tend to have E-values much
less than 1.0.
An E-value near 1.0, or even substantially larger than 1.0, does not necessarily mean that
the corresponding hit is biologically irrelevant; however, the larger the E-value, the
greater the chance that the similarity between the hit and the query is due to mere
coincidence.
E-values are calculated from the following three factors:
1. The bit score. Since a larger bit score is less likely to be obtained by chance than is a
smaller bit score, larger bit scores correspond to smaller E-values.
2. Length of the query. Since a particular bit score is more easily obtained by chance with
a longer query than with a shorter query, longer queries correspond to larger E-values.
3. Size of the database. Since a larger database makes a particular bit score more easily
obtained by chance, a larger database results in larger E-values. To see how E-values are
calculated, perform a "Compare Two Sequences" search with the following sequences:
acaagctcagaatttaatttgtgcgaagctcagcattaatcgtacttcatatagccctg
and
acaagctcagaatttaatatgtgcgttgctcagacattaatcacttcatatagccctg
Copy the alignment and relevant data into your answer sheet.
Above each alignment in the BLASTn report is the associated bit score (S) and, in
parentheses after the bit score, the raw score (R). For example, the first hit (E-value near
0.007) has S = 62.2 bits and R = 32. E-values are computed directly from bit scores,
which are in turn computed directly from raw scores.
Raw Scores
The raw score is calculated by counting the number of identities, mismatches, gaps, and
“-” characters in the alignment and setting R = aI + bX - cO - dG, where:
I is the number of identities in the alignment, and a is the reward for each identity
X is the number of mismatched nucleotides, and b is the “reward” for each mismatch
O is the number of gaps, and c is the penalty for opening a gap
G is the total number of “-” characters, and d is the penalty for each “-” in the gap
The values of the parameters a, b, c, and d are printed at the bottom of the BLASTn
report. The default values are a = 1, b = -3, c = 5, and d = 2; these values can be
changed on the “Other advanced” line. For example, in our first alignment,
there are 54 identities, 3 mismatches, 3 gaps, and 3 “-” characters. Calculate R by putting
these values into the equation above. Do you get the same number as reported (in
parenthesis beside the “score”?
Bit Scores
The bit score is obtained from the raw score using the equation
where  and K (also found somewhere on the Blast site—I just can't find everything
anymore!) are normalizing parameters. Assuming =1.37 and K=0.711, and using the R
value of 32, calculate the Bit score.
As a result of the normalizing process, bit scores and E-values are independent of the
scoring system, allowing those calculated with particular rewards and penalties to be
compared directly to those calculated with different rewards and penalties.
E-values
Finally, the E-value is calculated as E=mn2-S, where m is the effective length of the
query, and n is the effective length (total number of bases) of the database. (Effective
lengths are adjusted from the actual lengths to account for the fact that an alignment
cannot start near the end of the sequence, the so-called edge effect.) In our example,
m=34 (19 nucleotides fewer than the 53 bases submitted) and n= 3,743,317. Plugging
these numbers into the E-value equation gives E=0.007, as shown in the BLASTn report.
Why is the E value different when you put only a smaller number of bases into the
search, even though the bases are the same as some included in a larger sequence?
Test another sequence, as follows (you can copy numbers—the program knows not to
consider them):
TTAACACGTGTTGTTGGGAGTTCGAACCTCCCCCTTCCAGCCATCTCTTCTGTTGGTGCA
TAGCTTAATGGTAAAGCCCTGGTCTCCAAAACCAGTCACCTAGGTTCGATTCCTGGTGCA
TCAGCCAAATCTGTCTAAAATAGTCCACCATGACAGATTTCCTCTTTGATCCCCACGCAC
ATGTTAGGTTCGACAAGATCGATCTCGTACCAGAGGCTCAAAATCCTCGGTTCAAGGAGT
TGATCGAAAATACCAACCTGAACTACAAATACGATCCGAATGCTCCTCTCATCGTTGACA
CGACGAAGTACGGTCCTCGTTCATTCTACATGAACAATAAGAAGATCAAACGTACTGGCG
TCGAAATCAACATGACTGATGAGATGGTGGAGGAGCTCTACAAGTGCTCGACTGATGTCT
TGTACTTTGCTGAGCGATATTATTACATTCGTACTCTTGACCACGGCAAGATCAAGATCC
CTCTCCGTGACTATCAGAAATTCTGGCTCCGCATTTACGAGGTCCCTGAGATCCGTAACC
GCGTGTGGCTCGCTTGTCGTCAGTCAGCCAAGTCCACGACACTGACCGTTGAGATCATGC
ATCGAATGCTCTTCAATGAGGATTTCGAATACGTCATCCTGGCCAACAAGGGTAACACGG
CTCGTGAAATCTTCTCGCGTGTCCGTATGGCATATGAACAGCTTCCTCTCTGGATGCAGA
TCGGCGTGACCGAATGGAACAAGGGTTCTGTGAAGCTCGAGAACGATTCCCGTGTCTTCG
CTGCTGCTTCTGGTTCTGACTCTGTCCGCGGTTTCTCTCCCAACGAAGTTCTCCTCGACG
AAGCGGCATTCGTTCGTAATGATGAAGAGTTCATGGCCTCCGTGTTCCCGACCATCTCGT
CTGGTTCTAAGTCTCGTCTGACCCAGATCTCCACGCCGAATGGCCCTCGTGGGATCTTCC
ACCGAGATTACACTCGTGCGACCAAGGGTCTCAACAACTACTTCTCCTACAAGGTTCCAT
GGCACTTTGTTCCTGGCCGTGATGAGGAATGGAAGAAGAAGCAGATCGAAGATACCTCGC
TCATGCAGTTCAAGCAGGAGCAGGATTGTGACTTCATGGGCACTACCGACGGCCTCATCG
ATGGTATGGTTCTCGAGAGCATCCAGGACAATATTCAGGCCCCAATTCTTGTCAATGATG
AGGAACTGTCGGATCCAGCGTATGCAATCTTCGAACTCCCGAAGGAAGGACACTCGTATA
TCGTCACTGCCGACACCGGTGAAGGTAAAGGCAAGGACTCGAGCACATTCACCGTGTTTG
ACGTTTCGACTCGTCCGTTCGTCCAGGTTGCTTCCTACAAATCTAACCAGGTTTCCCAGC
TGATCTTCCCAAACAAGCTGGTCAAGATCGCAGAGACATACAATAATGCTCTCTTGATCC
CAGAAAGAAATAACACATCCGGCGGCACTGTCTGCTATAAGGTCTATTATGAACTCGAAT
ACCCGAACGTCTTCCTCCAGGGCGATGGCGAAATGGACATCGGCGTCCATGTCTCTCATG
CTGTCAGATCCCTCGGTGTCAATACCCTCCGTGGCCTCGTTGAAAAGGGCGGTCTCATTA
TCCGAGATGAACGGACCTTCAAGGAACTAGCGAACTTCCGTCTCCAGAAGAACGGGAAAT
ATGCTGCTCCAGAAGGTGAACATGATGATATGGTCCAGAACCTTTGGATATTCTCGTGGT
ATACGGCTGGTGACGAATTCGAAGAAGCGATGAAGGAAAATATCTACAATGACCTCTATC
GTGAAGAGCTCCAGAGCATCGAGAACCTGAAGGTCACCTCTGCGAACGACGCTTATGACC
CGTATAATGTGAAACCAGTGGCAGGAAAGGCGGCCTCTGCGTTTTGGTAAGAAAGGACAA
ATGATGGCATATGATCTATTCTCGGGTACCGTGGCCAATATCCCGGACCTGTCCACTCTC
AAAGAGAAGACGAGAGAGCTTCTCCAGATCCCTGTGTCGGCTGGCCTGGATATCACTCGC
TGGCTGAACGCACCTTCTCTCAATATCCCGGACCCGTTCAAGGAGTTCAATTTCAAATTC
AAGGGCGGTCGAGTATCGTTCGAGGAATATGAAAGGAATTTTCTTCCGAAAGTTCCTAAC
ATTCTCCAGTCGGCCAATCCGATCGGGTTCCAGGATGTCATCAGTGTCTTGGAGACGTTG
Perform a BLASTn analysis (NOT MEGABLAST). Note the color coding of the
matches. What is the best match? Now choose BLASTx, which compares translated
nucleotide sequences to protein sequences. What is the best match for this search? Why
are the results different?