Biomedical Engineering Reference
In-Depth Information
be created with the
sudophred
utility (see Protocol 2.4). Subsequently, the real resequenc-
ing traces can be assembled together with the reference using the
phrap
command (see
Protocol 2.5). Typically, assemblies are written to ACE (.ace) files, a format utilized by
many downstream analysis tools.
PROTOCOL 2.4 Creating Traces from the Reference Sequence
with the
sudophred
Utility
Equipment and reagents
Reference sequence for the target region in FASTA format.
•
The
sudophred
utility from the
Polyphred
package.
q
•
Method
1 Create or verify the existence of the
chromat_dir
and
phd_dir
subdirectories.
2Runthe
sudophred
tool as follows: sudophred [reference.fasta] -r
3 To specify a minimum base quality value for the reference, use the -q parameter:
sudophred [reference.fasta] -r -q 20
4 For further options, invoke the command: sudophred -h
5 Verify that reference trace files were created in the
chromat_dir
and
phd_dir
subdirectories.
Notes
q
Available from http://droog.gs.washington.edu/polyphred [19].
PROTOCOL 2.5 Assembling Reads on the Reference Sequence
with the
Phrap
program
Equipment and reagents
Reference sequence traces generated by
sudophred
(see Protocol 2.4)
•
•
Clone and subclone vector sequences in FASTA format for vector screening
•
Sequence files in trace or PHD format
The
Phred
/
Phrap
/
cross_match
programs
r
•
The
phredPhrap
script.
s
•
Method
1 Create the four directories expected by the
phredPhrap
program.
2 Move or copy the trace files into the
chromat_dir
subdirectory.
