This mode of assembly uses the Phrap program, developed by Phil Green. For best Phrap and Gap4 integration a modified version (gcphrap) is required. The main purpose of the change is to allow Phrap to support the Experiment File format for both input and output. For this version please email Phil Green (email@example.com).
A summary of the benefits of using the Gap4 Phrap interface follows.
Finally note that if Phred is used for base calling Gap4 will operate best with the "confidence" probability mode enabled. See section The Consensus Calculation.
To get the most out of Phrap (and Gap4) a base caller which generates confidence values should be used. The Phred base caller (also written by Phil Green) is probably the most widely used example and has been extensively tested in conjunction with Phrap.
There are two significant methods of running Phred. The first is to produce a `.phd' file containing the new base calls and confidence values. The second is to produce a new SCF file. For use with Gap4 we recommend outputting SCF files as this will ensure a correct synchronisation between the trace displays and the sequence displays. In the following example phred is used to reassign the base calls for all traces held in the `chromat_dir' directory, writing new SCF files into the `new_chromat_dir' directory.
phred -id chromat_dir -cd new_chromat_dir
These SCF files can then be passed into pregap in the same fashion as normal
except for one additional
.pregaprc parameter ("
disable Pregap's own quality value assignment. Add this to your
`.pregaprc' file using
echo "do_eba=No" >> .pregaprc. If
cross_match needs to be used, instead of the vector_clip program used in
pregap, the Experiment File patch also allows cross_match to read (but
currently not write) Experiment Files. This means that Pregap can be used with
vector clipping disabled to generate the Experiment Files. cross_match can
then be used to output clipping sequence in a fasta file which could be passed
cross_match fofn.passed vector.seq -minmatch 12 -minscore 20 -screen > scr.out
The above example uses cross_match to analyse the pregap output of files listed in `fofn.passed'. This will produce a new file named `fofn.passed.screen' which will be a Fasta format file rather than a new file of filenames. However this filename can be given to the Phrap interface in Gap4 instead of the requested file of filenames and Phrap will automatically detect that this is a fasta file.
The Phrap assemble command takes a file of Experiment File filenames and passes these into Phrap for assembly. The resulting assembly from Phrap is then automatically entered into the Gap4 database (implemented using the Directed Assembly command).
The "Destination directory" in the above dialogue is the location for Phrap to output the assembled data in Experiment File format. These files do not need to be kept unless further analysis of the assembly outside of Gap4 is required. Internally they are used as input to the Directed Assembly option.
If you have specific Phrap parameters add them to the "Other phrap parameters" entry box. Please see the documentation that came with Phrap for a list of available parameters. If in doubt, just leave this blank.
Next there is the option to perform quality clipping (see section Quality clipping) and difference clipping (see section Difference clipping). These options are useful for tidying up the Phrap assembly. To see the raw Phrap assembly turn both of these off. They may be selected from the Gap4 Edit menu at a later stage without the need to rerun phrap.
Pressing OK will then start Phrap running. At the end of assembly you should be presented with output in the main text window and the Contig Selector window. Phrap will also have produced several files named after the input file of filenames. These have extensions `.contigs', `.contigs.qual', `.log' and `.singlets'. The Phrap documentation explains their contents. The main output of Phrap is also written to disk as a file named `stdout', held in the destination directory.
Gap4 also provides a graphical interface for using Phrap to reassemble a set of sequences already held within a Gap4 database. It extracts readings from the database, reassembles them using Phrap, and enters the newly assembled readings back into the database.
The dialogue is identical to that used in the Phrap Assemble command. For dialogue help please see section Phrap Assembly.
Edits to both sequences and confidence values are preserved. Annotations are also preserved although they may have their length changed if the reassembly results in adding or removing a pad within the annotated segment.
Although it is not necessary to understand the individual steps taken during reassembly it is instructive and may answer some questions.
If you wish to use the new Phrap within your own scripts you will probably need to understand how to use Phrap on the command line. The full Phrap documentation should come with the Phrap distribution. Here we just give an outline of the changes involved in handling Experiment files.
Phrap automatically detects the file type for input sequences. If the contents of the file start with a '>' it is assumed to be a Fasta file and processing is identical to the previous Phrap version. Otherwise the file is assumed to be a file of Experiment File filenames.
With Experiment Files, the
CH line types are
used to hold information which Phrap normally requires in the reading name (in
a Phrap specific format). We produce a new sequence name for phrap consisting
//file_name where phrap_name is generated from
the aforementioned Experiment File lines. This allows for minimal Phrap source
changes whilst retaining complete user control over naming conventions. Phrap
also reads the
SR line types, which specify the vector
clips. Quality clip information is ignored.
-exp parameter is given to Phrap, Phrap reads the next argument
as a directory in which to write Experiment Files. Use "
-exp ." to
overwrite the input files, although this is not usually recommended. Without
this parameter Phrap will output fasta or ace format files in the normal
The filenames of the Experiment Files are the same as the input file names.
The Phrap reading name is processed to strip off the phrap_name
to obtain the original Experiment File name. This Experiment File is then read
and all relevant information copied out to the newly created Experiment File.
TG lines) have their positions and lengths updated as
required (due to padding). New quality left (
QL) and quality right
QR) line types created. Finally an Assembly Position (
is added. This provides the necessary information for the Gap4 Directed
Assembly option to enter the sequences.
One result of this method is that it is possible to use cross_match with a set of Experiment files to output a screened fasta file and then to run Phrap on the fasta file producing Experiment Files. Despite the fact that Phrap was only given a fasta file, the original Experiment File contents are used in writing out the aligned Experiment Files.