BIOBAR : USER MANUAL

BIOBAR : USER MANUAL
INSTALLING:
AUTOMATIC INSTALLATION
o Make sure Biobar.dotm is open and double-click the “Click to Install Biobar”
button in the document
MANUAL INSTALLATION
o The Biobar.dotm file needs to be placed in the STARTUP folder for Microsoft
Word so that the toolbar is available for every document.
XP -
C:\Documents and Settings\\Application
Data\Microsoft\Word\Startup
Vista –
C:\Users\\AppData\Roaming\Microsoft\Word\STARTUP
SELECTION:
OPTIONS:
Select the sequence(s) that you wish to perform an operation on
You should have two enters between sequences
o NOTE: if you are entering sequences in FASTA format (with a “>” denoting the
header), the “>” will be used to separate sequences, NOT two enters
Basic Options
o Default Output
Raw - your results will be displayed in the raw format
FASTA - your results will be displayed in the FASTA format
if a header was not provided, a numbered default one will be
created
GenBank – your results will be displayed in the GenBank format
o Default Output Location
Append to Document – your results will be printed beneath the selection
Create New Document – your results will be printed on a new Document
Save to Clipboard – your results will be saved to the Clipboard; use Paste
(Ctrl + V) to retrieve them
Replace Selection – your results will overwrite the selection
Advanced Options (Pop-up)

o % ATCG for DNA
This represents the percentage of A’s, T’s, C’s and G’s that must be in a
sequence for it to be considered DNA
NOTE: the percentage must be entered as an integer, so enter 70 for
70%, NOT 0.7
o Allow IUB Code
If checked, the following IUB characters are allowed in DNA sequences:
A, T, C, G, R, Y, M, K, S, W, H, B, V, D, N
Characters other than the above are filtered out
If not checked, the following characters are allowed in DNA sequences
A, T, C, G
Whether a sequence is DNA or Protein is determined by % ATCG
Characters other than A, T, C or G are filtered out
o If “ATGAAMGA” were selected, “ATGAAGA” would be
processed
o Pseudocounts
LaPlace: pseudocounts for the PSFM calculated using LaPlace’s method
10^-50: pseudocounts intended only to avoid 0 values for log
calculations
Translation Options
o Offset
Refers to the frame of translation (0, +1, +2)
o Genetic Code (GCode)
Denotes the genetic code used in translation and reverse translation
Standard
Vertebrate Mitochondrial
Yeast Mitochondrial
Mold, Protozoan and Coelenterate Mitochondrial/Mycoplasma
and Spiroplasma Mitochondrial
Invertebrate Mitochondrial
Ciliate, Dasycladacean and Hexamita Nuclear
Echinoderm and Flatworm Mitochondrial
Euplotid Mitochondrial
Bacterial, Archaeal and Plant Plasmid
NOTE: this option is overridden if a Codon Usage Table is entered
o Codon Usage Table
If not checked, the default Genetic Code selected will be used in reverse
translation. The following options are allowed:
Uniform - for each amino acid in the sequence(s), a random codon
will be chosen from those associated with that amino acid

IUB - for each amino acid in the sequence, its IUB code will be
used
If checked, user will be prompted to enter a Codon Usage Table after the
Reverse Translation button has been pressed, which will be used in
reverse translation. The following additional options are allowed:
Best - for each amino acid in the sequence(s), the codon with the
highest frequency associated with that amino acid (determined
by Codon Usage Table) will be selected
Random Best - for each amino acid in the sequence(s), a random
number will be generated and a codon associated with that amino
acid will be selected relative to its frequency (determined by
Codon Usage Table)
NOTE: Please use the codon usage tables found on
http://www.kazusa.or.jp/codon/ using the option for “a style like
CodonFrequency output in GCG”
DNA Statistics Options
o Window-Based % GC
Length – this value will serve as the sliding window length when
calculating % GC
Step-size – the value will serve as the number of base-pairs the sliding
window shifts each time (useful for long sequences)
o N-Gram
The user-entered value will serve as n (the length of each “gram”)
Only display N-Grams found in the sequence
With this option selected, only N-Grams with a nonzero count will
be displayed in the table.
o If ATAG were selected, with n = 2, only AT, TA, AG will be
displayed in the table rather than AA, AC, AG, AT,…,TT
Include the reverse complement
With this option selected, the counts of N-Grams of the reverse
complement will be added to the counts of the original sequence
o Molecular Weight
Consider DNA sequences double stranded
Include a 5’ triphosphate on RNA sequences
Include a 5’ monophosphate on DNA sequences
o Codon Usage Table
Offset
Refers to the frame (0, +1, +2)
Table Display

White-space-the table will be displayed in the same form as the
codon usage tables from http://www.kazusa.or.jp/codon/
Table-the table will be displayed in a Microsoft Word table
Substring Search Options
o ORF
Minimum codon length – defines the minimum length of an open reading
frame to be reported
o Optimize based on:
Length - when selected, longer ORFs will be ranked higher
CAI and Length – when selected, CAI scores are also taken into
consideration in ranking ORFs
o For Substring and Substring with Gap
Mismatch Threshold – only sequences with less mismatches than the
value entered will be displayed
Site Search
o Ri and Iseq Scoring Options
% GC of Genome– this value will be used to calculate the composition of
the genome sequence that the motif is scored upon
Motif – enter a motif of sequences, all the same length in either Raw or
FASTA format
o Dyad Pattern Search
% GC of Genome – this value will be used to calculate the composition of
the genome sequence that the motif is scored upon
Motif – enter a motif of sequences, all the same length in either Raw or
FASTA format
Threshold - each dyad found must have a score higher than a userdefined
constant times the information content of the motif to be
reported
Gap – defines the range of base-pairs as a spacer between two dyads
For second dyad:
Mirror Motif – when selected, the other half of the dyad will be the
reverse complement of the motif entered
Duplicate Motif – when selected, the other half of the dyad will be
the same as the motif entered
o Consensus Logo
% GC of Genome – this value will be used to calculate the composition of
the genome sequence that the motif is scored upon
Use IUB for equally probably bases – if this option is checked, IUB
characters will be used for two bases that occur at equal frequencies in a
position in the motif. For example, if “A” and “G” are equally likely, “R”
will be used for that position

Motif Discovery
o Gibbs Sampling
% GC of Genome – this value will be used to calculate the composition of
the genome sequence that the motif is scored upon
Window Length – this value determines the length of the window
sampled by Gibbs Sampling
Number of Iterations – the number of times the program should cycle
before reporting results (higher number of iterations means slower, but
more accurate results)
o Greedy Search
% GC of Genome – this value will be used to calculate the composition of
the genome sequence that the motif is scored upon
Window Length – this value determines the length of the window
sampled by Gibbs Sampling
Number of Iterations – the number of times the program should cycle
before reporting results (higher number of iterations means slower, but
more accurate results)
o Dyad Motif Search
Dyad Length – if values 5 ± 1 were entered, dyads of length 4, 5, and 6
will be considered
Spacer Length – if values 5 ± 1 were entered, spacers of length 4, 5, and 6
will be allowed
Mismatch Threshold – determines the maximum number of mismatches
that should be allowed between the two dyads
Palindrome – the dyads must be reverse complements of each other
Direct Repeat – the dyads must be identical
Pair-wise Alignment
o Scoring Options
The first selection allows the user to define constant values to be used to
score matches and mismatches
The second selection allows the user to define more specific values if
they would like to weigh certain mismatches/matches as less or more
important than another
NOTE: if protein sequences are selected, BLOSUM62 will be used to score
the matches and mismatches between the sequences
o GEP – this value defines the gap extension penalty
o GOP – this value defines the gap opening penalty
o Limit results to X alignments – in the case of split paths, the program will report
no more than X sequences

FUNCTIONALITY :
Basic Manipulation
o Convert Sequence To:
Raw – The sequence(s) selected will be printed in the raw format
FASTA – The sequence(s) selected will be printed in FASTA format
A default header (> # Default) will be created if one is not
provided (> # [user input])
GenBank – The sequence(s) selected will be printed in GenBank format
o Reverse
DNA, RNA and protein sequences can be reversed
ATAGTAGAT -> TAGATGATA
o Complement
Only DNA and RNA sequences may be complemented. An error message
will be displayed if a protein sequence is selected. If DNA, RNA, and
protein sequences are selected, an error message will be displayed and
only the DNA and RNA sequence(s) will be complemented. IUB
characters will be ignored.
ATAGTAGAT -> TATCATCTA
o Reverse Complement
Only DNA and RNA sequences may be complemented. An error message
will be displayed if a protein sequence is selected. If DNA, RNA and
protein sequences are selected, an error message will be displayed and
only the DNA and RNA sequence(s) will be reverse complemented. IUB
characters will be ignored.
ATAGTAGAT -> ATCTACTAT
Translation
o Forward (Translation)
Only DNA sequences may be translated. An error message will be
displayed if a protein sequence is selected. If DNA, RNA and protein
sequences are selected, an error message will be displayed and only the
DNA sequence(s) will be translated.
If IUB is selected:
Due to possible duplicities with IUB code, a list of all codons
corresponding to that IUB code will be generated and a random
one will be chosen to be used in translation
o Example:
GCode = Standard:
TGB -> [TGC, TGT, TGG] -> C or W

If an incorrect character (not A, T, C, G, R, Y, M, K, S, W, H, B, V, D,
N) is found, an error message will be displayed and translation
will not occur
Translation will be performed according to the default Genetic Code
selected and according to the offset specified in Translation Options
NOTE: additional bases will be removed
Examples: (italicized characters are ignored)
Offset = 0, GCode = Standard: ATAGTAGAT -> IVD
Offset =1, GCode = Standard: ATAGTAGAT -> **
o Reverse (Translation)
Only protein sequences may be reverse translated. An error message
will be displayed if a DNA or RNA sequence is selected. If DNA, RNA and
protein sequences are selected, an error message will be displayed and
only the protein sequence(s) will be reverse translated.
Reverse Translation will be performed according to the method specified
in the Reverse Translation/Translation Options
If the user provides a Codon Usage Table, the reverse translation
will occur according to the Codon Usage Table rather than the
default Genetic Code
Examples:
o GCode = Standard: IVD -> ATAGTAGAT
o (Translation) Map
Only DNA sequences can be used to create a translation map. An error
message will be displayed if a protein or RNA sequence is selected. If
DNA, RNA, and protein sequences are selected, an error message will be
displayed and translation maps will only be displayed for the DNA
sequence(s). The sequence(s) is (are) translated for each of the frames
and all of the resultant sequences are displayed together.
If IUB is selected:
Due to possible duplicities with IUB code, a list of all codons
corresponding to that IUB code will be generated and a random
one will be chosen to be used in translation
o Example:
GCode = Standard:
TGB -> [TGC, TGT, TGG] -> C or W
If an incorrect character (not A, T, C, G, R, Y, M, K, S, W, H, B, V, D,
N) is found, an error message will be displayed and translation
will not occur
DNA Sequence Statistics
o Global %GC

Only DNA sequences can be used.. An error message will be displayed if
a protein or RNA sequence is selected. If both DNA and protein
sequences are selected, an error message will be displayed and %GC will
only be calculated for the DNA sequence(s).
Calculates the %GC content for the entire sequence and displays it in a
table.
o Window %GC
Only DNA sequences can be used.. An error message will be displayed if
a protein or RNA sequence is selected. If both DNA and protein
sequences are selected, an error message will be displayed and %GC will
only be calculated for the DNA sequence(s).
Calculates the %GC content for windows of length defined by user in the
Sequence Statistics Options and displays them in a table, indexed by
position.
o Nucleotide Frequencies
Only DNA sequences can be used. An error message will be displayed if a
protein or RNA sequence is selected. If both DNA and protein sequences
are selected, an error message will be displayed and nucleotide
frequencies will only be calculated for the DNA sequence(s).
Calculates frequency of each nucleotide for the entire sequence and
displays it in a table.
o N-Gram
Only DNA sequences can be used. An error message will be displayed if
a protein or RNA sequence is selected. If sequences of all types are
selected, the N-Grams will only be calculated for the DNA sequence(s).
IUB characters will be ignored
Takes the value of N from Sequence Statistics Options and generates a list
of all possible “words” of length N (from the characters A, T, C, G) and
their counts within the sequence.
See Sequence Statistics Options for other options relating to N-Grams
o Codon Usage Table
Only DNA sequences can be used. An error message will be displayed if
a protein or RNA is selected. If sequences of all types are selected, the
codon usage table will only be generated for the DNA sequence(s). IUB
characters will be ignored
Generates a codon usage table (using the offset from Sequence Statistics
Options) using the sequence(s) that are selected.
See Sequence Statistics Options for options relating to table display
o Calculate Molecular Weight
Open to DNA, RNA and protein sequences.

See Sequence Statistics Options for options relating to Molecular Weight
Protein Sequence Statistics
o GRAVY
GRAVY can only be calculated for protein sequences. An error message
will be displayed if a DNA or RNA sequence is selected. If sequences of all
types are selected, GRAVY will only be calculated for protein
sequence(s).
GRAVY is the average of the hydropathicites of the amino acids.
o Isoelectric Point
Isoelectric points can only be calculated for protein sequences. An error
message will be displayed if a DNA or RNA sequence is selected. If
sequences of all types are selected, the isoelectric point will only be
generated for protein sequence(s).
The isoelectric point is the pH at which the protein carries a zero net
electric charge.
o Generate Protein Statistics
Protein statistics can only be calculated for protein sequences. An error
message will be displayed if a DNA or RNA sequence is selected. If
sequences of all types are selected, the protein statistics will only be
generated for the strict DNA sequence(s).
Protein statistics provides a count and percentage of each of the amino
acids for each sequence.
Substring Search
o Find ORFs (Open Reading Frames)
ORFs can only be found for DNA sequences. An error message will be
displayed if a protein or RNA sequence is selected. If sequences of all
types are selected, the ORFs will only be found for the DNA sequence(s).
ATG, TTG, CTG and GTG are all considered start codons
Different Output Locations:
BELOW – Only prints the top-scoring ORF
REPLACE – Highlights the top-scoring ORF
CLIPBOARD – Saves all discovered ORFs
NEW DOC – Prints all discovered ORFs
See Motif Discovery Options for options relating to ORFs
o Find Substring
Substring searches can only be completed for DNA sequences. An error
message will be displayed if a protein or RNA sequence is selected. If
sequences of all types are selected, the substring search will only be
completed for the DNA sequence(s).

IUB characters are considered fractional mismatches depending on
which DNA bases they stand for. For example, B is considered to be 1/3
T, 1/3 G and 1/3 C.
Find Substring with Gap allows the user to search for two substrings
separated by a gap of defined length.
Different Output Locations
BELOW, NEW DOC and CLIPBOARD
o Returns all results
REPLACE
o Highlights the matches in the sequence with a darker color
representing a better scoring sequence
Site Search
o Search (Ri sequence and I sequence)
These Site Search mechanisms can only be completed for DNA sequences.
An error message will be displayed if a protein or RNA sequence is
selected. If sequences of all types are selected, the site search will only
be completed for the DNA sequence(s).
In the motif, only DNA sequences may be entered.
Reports the Ri Score or Iseq Score
o Dyad Pattern Search
Dyad Pattern Search can only be completed for DNA sequences. An error
message will be displayed if a protein or RNA sequence is selected. If
sequences of all types are selected, the site search will only be completed
for the DNA sequence(s).
In the motif, only DNA sequences may be entered.
Different Output Locations:
BELOW, NEW DOC and CLIPBOARD
o Returns all results
REPLACE
o Highlights the dyads in the sequence with a darker color
representing a better scoring sequence
Motif Discovery
o Gibbs Sampling and Greedy Search
Gibbs Sampling can only be completed for DNA sequences. An error
message will be displayed if a protein or RNA sequence is selected. If
sequences of all types are selected, the sampling will only be completed
for the DNA sequence(s).
The program will complete the number of iterations requested 10 times
and return the motif that has the greatest IC content
Different Output Locations:
BELOW, NEW DOC and CLIPBOARD

o Returns the best result
REPLACE
o Highlights the motif in each sequence
o Dyad Motif Search
The Dyad Motif Search can only be completed for DNA sequences. An
error message will be displayed if a protein or RNA sequence is selected.
If sequences of all types are selected, the motif search will only be
completed for the DNA sequence(s).
o Consensus Logo
The consensus logo can only be generated with a motif of DNA sequences
The motif to generate a pseudo-consensus logo can be selected in the
document or entered into a pop-up input box when the button is clicked.
Information Content will be calculated using R sequence or Relative
Entropy, depending on which is selected in the Advanced Options
The text is likely to be small, and can be scaled up or down using the
arrows in the Resources Group (though resolution may be lost)
Pair-Wise Alignment
o Needleman-Wuncsh and Smith-Watermann
Pair-wise alignment can be performed with both DNA and Protein
sequences.
Protein sequences are scored using the BLOSUM62 matrix and DNA
sequences are scored using user-defined values.