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2021-12-04 13:07:58 +08:00
@-1. TX 0 @General
@-2. T 0 @Screen control
@-2. X 0 @Screen
@-3. TX 0 @Modification
@0. TX -1 @SAP
This is an interactive program whose primary use is for
managing shotgun sequencing projects, but it can also be used for
handling alignments of other sequences, including those of proteins.
Currently the maximum 'gel reading' length is set to 4096
characters. Almost all of the information below describes the use of
the program for shotgun projects, but those using the programs for
handling other sequence alignments should interpret it accordingly.
The data for such a project is stored in a special type of database.
The program contains the tools that are required to type in gel
readings, screen them against vector sequences and restriction
sites; enter new gel readings into the database (automatically
comparing and aligning them). In addition it contains editors and
functions to examine the quality of the aligned sequences.
There are three main menus: "general", "graphics" and
"modification", and some functions have submenus.
The general menu contains the following options:
0 = List of menus
? = Help
! = Quit
3 = Open a database
4 = Edit contig
5 = Display a contig
6 = List a text file
7 = Direct output to disk
8 = Calculate a consensus
17 = Screen against restriction enzymes
18 = Screen against vector
19 = Check consistency
25 = Show relationships
27 = set parameters
28 = Highlight disagreements
29 = Examine quality
The graphics menu contains:
0 = List of menus
? = Help
! = Quit
10 = Clear graphics
11 = Clear text
12 = Draw ruler
13 = Use cross hair
14 = Change margins
15 = Label diagram
16 = Plot map
33 = Plot single contig
34 = Plot all contigs
The modification menu contains:
0 = List of menus
? = Help
! = Quit
4 = Edit a contig
9 = Screen edit
20 = Auto assemble
21 = Enter new gel reading
22 = Join contigs
23 = Complement a contig
24 = Copy database
26 = Alter relationships
30 = Auto edit a contig
31 = Type in gel readings
32 = Extract gel readings
The enter new gel reading menu contains:
? = Help
! = Quit
3 = Complete entry
4 = Edit contig...
5 = Display overlap
6 = Edit new gel reading...
The join contig menu contains:
? = Help
! = Quit
3 = Complete join
4 = Edit left contig...
5 = Display joint
6 = Edit right contig...
7 = Move join
The alter relationships menu contains:
? = Help
! = Quit
3 = Line change
4 = Edit single gel reading...
5 = Delete contig
6 = Shift
7 = Move gel reading
8 = Rename gel reading
9 = Break contig
The edit menu contains:
? = Help
! = Quit
3 = Insert
4 = Delete
5 = Change
Overview of the methodology
The shotgun sequencing strategy
In the shotgun sequencing procedure the sequence to be
determined is randomly broken into fragments of about 400
nucleotides in length. These fragments are cloned and then selected
randomly and their sequences determined. The relationship
between any pair of fragments is not known beforehand but is
found by comparing their sequences. If the sequence of one
found to be wholly or partially contained within that of another
for sufficient length to distinguish an overlap from a repeat
then those two fragments can be joined. The process of select,
sequence and compare is continued until the whole of the DNA to
be sequenced is in one continuous well determined piece.
Definition of a contig
A CONTIG is a set of gel readings that are related to
one another by overlap of their sequences. All gel readings
belong to a contig and each contig contains at least one gel
reading. The gel readings in a contig can be summed to produce a
continuous consensus sequence and the length of this sequence is the
length of the contig. The rules used to perform this summation are
given under "the consensus algorithm". At any stage of a
sequencing project the data will comprise a number of contigs; when
a project is complete there should be only one contig and its
consensus will be the finished sequence. Note that since being
introduced and defined as above the word "contig" has been taken up
by those involved in genomic mapping. In that context the consensus
with a precise length is not defined.
Introduction to the computer method
It is useful to consider the objectives of a sequencing
project before outlining how we use the computer to help achieve
them. The aim of a shotgun sequencing project is to produce an
accurate consensus sequence from many overlapping gel readings. It
is necessary to know, particularly at the latter stages of the
project, how accurate the consensus sequence is. This enables us to
know which regions of the sequence require further work and also to
know when the project is finished. To show the quality of the
consensus, the programs described here produce displays like that
shown below.
10 20 30 40 50
-6 HINW.010 GCGACGGTCTCGGCACAAAGCCGCTGCGGCGCACCTACCCTTCTCTTATA
CONSENSUS GCGACGGTCTCGGCACAAAGCCGCTGCGGCGCACCTACCCTTCTCTTATA
60 70 80 90 100
-6 HINW.010 CACAAGCGAGCGAGTGGGGCACGGTGACGTGGTCACGCCGCGGACACGTC
-3 HINW.007 GGCACA*GTC
CONSENSUS CACAAGCGAGCGAGTGGGGCACGGTGACGTGGTCACGCCG-G-ACA-GTC
110 120 130 140 150
-6 HINW.010 GATTAGGAGACGAACTGGGGCG3CGCC*GCTGCTGTGGCAGCGACCGTCG
-3 HINW.007 GATTAG4AGACGAACTGGGGCGACGCCCG*TGCTGTGGCAGCGACCGTCG
-5 HINW.009 GGCAGCGACCGTCG
17 HINW.999 AGCGACCGTCG
CONSENSUS GATTAGGAGACGAACTGGGGCGACGCC-G-TGCTGTGGCAGCGACCGTCG
160 170 180 190 200
-6 HINW.010 TCT*GAGCAGTGTGGGCGCTG*CCGGGCTCGGAGGGCATGAAGTAGAGC*
-3 HINW.007 TCT*GAGCAGTGTGGGCGCTGC*CGGGCTCGGAGGGCATGAAGTAGAGC*
-5 HINW.009 TCT*GAGCAGTGTGGGCG*T*G*CGGGCTCGGAGGGCATGAAGTAGAGC*
17 HINW.999 TCTCGAGCAGTGTGGGCGCTG**CGGGCTCGGAGGGCATGAAGTAGAGCG
12 HINW.017 GTAGAGC*
CONSENSUS TCT*GAGCAGTGTGGGCGCTG-*CGGGCTCGGAGGGCATGAAGTAGAGC*
This is an example showing the left end of a contig from
position 1 to 200. Overlapping this region are gel readings
numbered 6, 3, 5, 17 and 12; 6, 3 and 5 are in reverse orientation
to their original reading (denoted by a minus sign). Each gel
reading also has a name (eg HINW.010). It can be seen that in a
number of places the sequences contain characters other than A,C,G
and T. Some of these extra characters have been used by the
sequencer to indicate regions of uncertainty in the initial
interpretation of the gel reading, but the asterisks (*) have been
inserted by the automatic assembly function in order to align the
sequences. Underneath each 50 character block of gel reading
sequences is the consensus derived from the sequences aligned above
(the line labelled CONSENSUS). For most of its length the consensus
has a definite nucleotide assignment but in a few positions there is
insufficient agreement between the gel readings and so a dash (-)
appears in the sequence. This display contains all the evidence
needed to assess the quality of the consensus: the number of times
the sequence has been determined on each strand of the DNA, and the
individual nucleotide assignments given for each gel reading.
So the aim is to produce the consensus sequence and, equally
important, a display of the experimental results from which it was
derived.
In order to achieve this the following operations need to be
performed:
1) Interpret autoradiographs and put individual gel readings into
the computer.
2) Check each gel reading to make sure it is not simply part of one
of the vectors used to clone the sequence.
3) Check each gel reading to make sure that those fragments that
span the ligation point used prior to sonication are not assembled
as single sequences.
4) Compare all the remaining gel readings with one another to
assemble them to produce the consensus sequence.
5) Check the quality of the consensus and edit the sequences.
6) When all the consensus is sufficiently well determined, produce a
copy of it for processing by other analysis programs.
It is very unlikely that this procedure will only be passed
through once. Usually steps 1 to 5 are cycled through repeatedly,
with step 4 just adding new sequences to those already assembled.
Generally step 6 is also used in order to analyse imperfect sequence
to check if it is the one the project intended to sequence, or to
look for interesting features. Analysis of the consensus, such as
searches for protein coding regions, can also help to find errors in
the sequence. The display of the overlapping gel readings shown
above can be used to indicate, not only the poorly determined
regions, but also which clones should be resequenced to resolve
ambiguities, or those which can usefully be extended or sequenced in
the reverse direction, to cover difficult regions.
The original individual gel readings for a sequencing project
are each stored in separate files. As the gel readings are entered
into the computer (usually in batches, say 10 from a film), the file
names they are given are stored in a further file, called a file of
file names. Files of file names enable gel readings to be processed
in batches.
For each sequencing project we start a project database. This
database has a structure specifically designed for dealing with
shotgun sequence data. In order to arrive at the final consensus
sequence many operations will be performed on the sequence data.
Individual fragments must be sequenced and compared in both senses
(i.e. both orientations) with all the other sequences. When an
overlap between a new gel reading and a contig are found they must
be aligned and the new gel reading added to the contig. If a new gel
reading overlaps two contigs they must be aligned and joined. Before
the two contigs are joined one of them may need to be turned around
(reversed and complemented) so they are both in in the same
orientation.
Clearly, keeping track of all these manipulations is quite
complicated, and to be able to perform the operations quickly
requires careful choice of data structure and algorithms. For these
reasons it is not practicable to store the gel readings aligned as
shown in the display above. Rather, it is more convenient to store
the sequences unassembled, and to record sufficient information for
programs to assemble them during processing. The data used to
assemble the sequences is called relational information.
The database comprises three files and they are described
under the section entitled "open database".
Before entry into the project database each new gel reading
must be compared to look for overlaps with all the data already
contained within the database. This last point is important: all
searching for overlaps is between individual new gel readings and
the data already in the database. There is no searching for overlaps
between sequences within the database; overlaps must be found before
new gel readings are entered into the database.
Below I give an introduction to how the sequencess are
processed by being passed from one function to the next.
This program is used to start a database for the project and
then the following procedure is used.
Data in the form of individual gel readings are entered into
the computer and stored in separate files using either program this
program or the digitizer program. Batches of these gel readings are
passed to the screening functions in this program to search for
overlaps with vector sequences ("screen against vector") or for
matches to restriction enzyme sites that should not be present
("screen against enzymes"). Each run of these screening functions
passes on only those gel readings that do not contain unwanted
sequences. Sequences are passed via files of file names and
eventually are processed by the automatic assembly function ("auto
assemble"). This function compares each gel reading with a consensus
of all the previous gel readings stored in the database. If it
finds any overlaps it aligns the overlapping sequences by inserting
padding characters, and then adds the new gel reading to the
database. Gels that overlap are added to existing contigs and gels
that do not overlap any data in the database start new contigs. If a
new gel overlaps two contigs they are joined. Any gel readings that
appear to overlap but which cannot be aligned sufficiently well are
not entered and have their names written to a file of failed gel
reading names.
Generally data is entered into the database in batches as just
described. The program is also used to examine the data in the
database, to enter gel readings that the automatic assembly function
cannot align ("enter new gel reading"), and to make final edits.
Edits to whole contigs can be made in several ways. An automatic
editor ("auto edit") will perform almost all edits without any user
intervention, but the program also gives access to the system editor
(EDT on the VAX), through the function "screen edit", and to simple
command driven editors ("edit contig" and "edit new gel reading").
Disagreements between gel readings in contigs and their consensus
sequences can be highlighted by use of the function "highlight
disagreements".
Editing the sequences is obviously an essential part of
managing a sequencing project. Editing is required when new
sequences are added, when contigs are joined, and when sequences are
corrected. A basic part of the strategy used here is that new gel
readings should be correctly aligned throughout their whole length
when they are entered into the database, and that when contigs are
joined they are edited so that they are well aligned in the region
of overlap. Alignment can be achieved by adding padding characters
to the sequences, and this is the way "auto assemble" operates when
adding new sequences to the database.
In order to search for overlaps that may have been missed due
to errors in the gel readings, the function "extract gel readings"
can be used to take copies of the gel readings at the ends of
contigs, and write them out as separate files. These can then be
compared with the database consensus using the "auto assemble"
function in a mode that forbids entry of data into the database, and
any gel reading matching two contigs will indicate a join that has
been missed. The joins can then be made interactively using "join
contigs". Missed matches can be found at this stage because the
errors in the sequences may have been corrected by new data.
Generally the users need not concern themselves with how the
relational information is used by the program, but it is necessary
to know how contigs are identified. Because contigs are constantly
being changed and reordered the program identifies them by the
numbers of the gel readings they contain. Whenever users need to
identify a contig they need only know the number or name of one of
the gel readings it contains. Whenever the program asks users to
identify a contig or gel reading they can type its number or its
archive name. If they type its archive name they must precede the
name by a slash "/" symbol to denote that it is a name rather than a
number. E.g if the archive name is fred.gel with number 99, users
should type /fred.gel or 99 when asked to identify the contig.
Generally, when it asks for the gel reading to be identified, the
program will offer the user a default name, and if the user types
only return, that contig will be accessed. When a database is opened
the default contig will be the longest one, but if another is
accessed, it will subsequently become the current default.
Further information is located in the following places. The
database files are described under "open database". The format for
vector and consensus sequences is given under "calculate a
consensus", as are the uncertainty codes used in gel readings.
The only program, other than this, relevant to sequencing is
the digitizer program and it is outlined briefly below.
The digitiser program is used for the initial input of gel
readings and for writing a file of file names. The program uses a
digitizer for data entry. A digitizer is a two dimensional
surface such as a light box which is such that if a special pen is
pressed onto it, the pens coordinates are recorded by a computer.
These coordinates can be interpreted by a program.
In order to read an autoradiograph placed on the light box the
user need only define the bottom of the four sequencing lanes and
the bases to which they correspond and then use the pen to point
to each successive band progressing up the gel. The program
examines the coordinates of each pen position to see in which of the
four lanes it lies and assigns the corresponding base to be
stored in the computer. Each time the pen tip is depressed to point
to a position on the surface of the digitizer the program sounds
the bell on the terminal to indicate to the user that a point has
been recorded. As the sequence is read the program displays it on
the screen.
@17. TX 1 @Screen against restriction enzymes
Used to compare gel readings against any restriction enzyme
recognition sequences that may have been used during cloning and
which should not be present in the data. Works on single gel
readings or processes batches accessed through files of file names.
The algorithm looks for exact matches to recognition sequences
stored in a file.
The file containing the recognition sequences must be
identified. The user must choose between employing a file of file
names, or typing in the names of individual gel reading files. If a
file of file names is used the program will also create a new file
of file names. When the option has finished operating this new file
will contain the names of all those gel readings that did not match
any of the recognition sequences. Hence it can be used for further
processing of the batch. The recognition sequences should be stored
in a simple text file with one recognition sequence per line.
@18. TX 1 @Screen against vector
Used to compare gel readings against any vector sequences that
may have been picked up during cloning. Works on single gel readings
or processes batches accessed through files of file names. The
algorithm looks for exact matches of length "minimum match length"
and displays the overlapping sequences.
The file containing the vector sequence must be identified.
The user must choose between employing a file of file names, or
typing in the names of individual gel reading files. If a file of
file names is used the program will also create a new file of file
names. When the option has finished operating this new file will
contain the names of all those gel readings that did not match the
vector sequence. Hence it can be used for further processing of the
batch.The vector sequence should be stored in a simple text file
with up to 80 characters of data per line. More than one vector can
be stored in a single file. If so each should be preceded by a 20
character title of the form <---m13mp8.001-----> where the < and >
signs and the number like .001 are obligatory. The number must be
preceded by a dot (.) and be 3 digits long. The total sequence in
the file must be < 50,001 characters in length.
@20. TX 2 @Auto assemble
Compares gel readings against the current contents of the
database and produces alignments. In its normal mode of operation
("entry permitted"), the function will automatically enter the gel
readings into the database, but if entry is not permitted it will
only produce alignments. It works on single gel readings or
processes batches of gel readings accessed through files of file
names. It is the usual way to enter data into the database.
The function will check the database for logical consistency
and will only procede if it is OK. Choose if gel readings should be
entered into the database, or if they should only be compared.
Choose between using a file of file names or typing file names on
the keyboard. If so selected, supply the file of file names. Also
supply a file of file names to contain the names of all the gel
readings that fail to get entered. Select the entry mode. Normal
assembly is appropriate for all but special cases, as is "permit
joins". Uses for the other modes are not documented here. Define a
minimum initial match length. Define a minimum alignment block (the
default value is taken in all but exceptional circumstances). Define
the maximum number of paddding characters allowed to be used in each
gel reading to help achieve alignment, and the same for the number
allowed in the contig for each gel reading. Finally define the
maximum percentage mismatch to be allowed for any gel reading to be
entered into the database. If for any gel reading, either of these
last three values is exceeded the gel reading will not be entered
into the database.
In operation the function takes a batch of gel readings
(probably passed on as a file of file names from one of the
screening routines) and enters them into a database for a sequencing
project. It takes each gel reading in turn, compares it with the
current consensus for the database, it then produces an alignment
for any regions of the consensus it overlaps; if this
alignment is sufficiently good it then edits both the new gel
reading and the sequences it overlaps and adds the new gel
reading to the database. The program then updates the consensus
accordingly and carries on to the next gel reading.
All alignments are displayed and any gel readings that do
match but that cannot be aligned sufficiently well have their names
written to a file of failed gel reading names. The function works
without any user intervention and can process any number of gel
readings in a single run. Those gel readings that fail can be
recompared using the same function (to find the current overlap
position) and the user can enter them into the database manually
using the "enter new gel reading" option.
Typical dialogue and output from the function is shown below.
(Note that output for gel readings 2 - 9 has been deleted to save
space).
Automatic sequence assembler
Database is logically consistent
? (y/n) (y) Permit entry
? (y/n) (y) Use file of file names
? File of gel reading names=demo.nam
? File for names of failures=demo.fail
Select entry mode
X 1 Perform normal shotgun assembly
2 Put all sequences in one contig
3 Put all sequences in new contigs
? Selection (1-3) (1) =
? (y/n) (y) Permit joins
? Minimum initial match (12-4097) (15) =
? Minimum alignment block (2-5) (3) =
? Maximum pads per gel (0-25) (8) =
? Maximum pads per gel in contig (0-25) (8) =
? Maximum percent mismatch after alignment (0.00-15.00) (8.00) =
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Processing 1 in batch
Gel reading name=HINW.004
Gel reading length= 283
Searching for overlaps
Strand 1
Strand 2
No matches found
Total matches found 1
Padding in contig= 0 and in gel= 1
Percentage mismatch after alignment = 1.8
Best alignment found
1 11 21 31 41 51
TTTTCCAGCG TGCGTCTGAC GCTGTCTTGC TTAATGATCT CCATCGTGTG CCTAGGTCTG
********** ********** ********** ********** ********** **********
TTTTCCAGCG TGCGTCTGAC GCTGTCTTGC TTAATGATCT CCATCGTGTG CCTAGGTCTG
1 11 21 31 41 51
61 71 81 91 101 111
TTGCGTTGGG CCGAGCCCAA CTTTCCCAAA AACGTATGGA TCTTACTGAC GTACA-GTTG
********** ********** ********** ********** ********** ***** ****
TTGCGTTGGG CCGAGCCCAA CTTTCCCAAA AACGTATGGA TCTTACTGAC GTACACGTTG
61 71 81 91 101 111
121 131 141 151 161 171
CTTACCAGCG TGGCTGTCAC GGCGTCAGGC TTCCACTTTA GTCATCGTTC AGTCATTTAT
********** ********** ********** ********** ********** **********
CTTACCAGCG TGGCTGTCAC GGCGTCAGGC TTCCACTTTA GTCATCGTTC AGTCATTTAT
121 131 141 151 161 171
181 191 201 211 221 231
GCCATGGTGG CCACAGTGAC G-TATTTTGT TTCCTCACGC TCGCTACGTA TCTGTTTGCC
********** ********** * ******** ********** ********** **********
GCCATGGTGG CCACAGTGAC GCTATTTTGT TTCCTCACGC TCGCTACGTA TCTGTTTGCC
181 191 201 211 221 231
241 251 261 271 281
CGCG--GTGG AATTACAGCG TTCCCTATTG ACGGGCGCAT CCAC
**** **** ********** ** * ***** ********** ****
CGCGACGTGG AATTACAGCG TT,CDTATTG ACGGGCGCAT CCAC
241 251 261 271 281
Batch finished
9 sequences processed
0 sequences entered into database
0 joins made
Note that "auto assemble" cannot align protein sequences.
@28. TX 1 @Highlight disagreements
Used in the latter stages of a project to highlight
disagreements between individual gel readings and their consensus
sequences. Characters that agree with the consensus are shown as :
symbols for the plus strand and . for the minus strand. Characters
that disagree with the consensus are left unchanged and so stand out
clearly. The results of this analysis are written to a file.
Before selecting this option create a file of the display of
the contig to be "highlighted". The option will ask for the name of
this file. Select symbols to denote "agreeing" characters on each
strand, the defaults are : and ., but any others can be used. Supply
the name of a file in which to put the output.
The display file needed as input for this option is created by
selecting "Redirect output", followed immediately by "display
contig", and then "Redirect output" again. The cutoff score used in
the consensus calculation can be set by option "set display
parameters". Note that for the highlight function there is a limit
of 50 for the number of gel readings that are aligned at any
position - ie the contig must be less than 51 gel readings deep at
its thickest point. I hope that those performing shotgun sequencing
never reach this limit, but those using the program for comparing
sequence families might.
Typical output from this function is shown below.
210 220 230 240 250
1 HINW.004 :C::::::::::::::::::::::::::::::::::::::::::AC::::
7 HINW.018 :*::::::::::::::::::::::::::::::::::::::::::CA::::
-4 HINW.017 ...............AC....
G-TATTTTGTTTCCTCACGCTCGCTACGTATCTGTTTGCCCGCG--GTGG
260 270 280 290 300
1 HINW.004 ::::::::::::*:D:::::::::::::::::::
7 HINW.018 ::::::::::::::::::::CA:::::T:*:::*::::::::::::CA:
-4 HINW.017 ..............................................A...
3 HINW.009 :::::::::::::::V::::::::::::::::::::::::::::*AV:::
-6 HINW.028 ......................A...
AATTACAGCGTTCCCTATTGACGGGCGCATCCACGCTGATTCTCTT-CTG
@32. TX 3 @Extract gel readings
Used to make copies of the aligned gel readings in a database,
to write them into separate files, and to write a corresponding file
of file names. It operates in two modes: either all gel readings are
extracted, or only those at the ends of contigs.
Choose which mode of operation is required and supply a file
of file names.
The gel readings are given their original names. If used to
extract the gel readings from the ends of contigs the function is
useful for checking for missed contig joins: the file of file names
can be used with the auto assemble function to recompare these gel
readings, and each should only overlap one contig. Any that overlap
two contigs will identify possible joins.
If the option is used to extract all the gel readings from a
database, a subsequent run of "auto assemble" can reconstitute a
database which has been corrupted. This rarely occurs and is
usually necessesitated by a user employing "alter relationships"
incorrectly without first having made a copy.
@1. TX 0 @Help
Help is available on the following topics :
@2. TX 0 Quit
This command stops the program and is the only safe way to
terminate a run of the program that has altered the contents of the
database in any way.
@3. TX 1 @Open a database
Opens existing databases or allows new ones to be started. The
function is automatically called into operation when the program is
started but can also be selected from the general menu.
Choose to open an existing database or start a new one, or if
! is typed when the program is first started, enter the program
without opening a database. Supply a project database name, and if
it already exists, the "version". If starting a new database define
the database size and if it is for DNA or protein sequences. The
database size is an initial size for the database. It can be
increased later during the project. It is the sum of the number of
gel readings plus the number of contigs.
Database names can have from one to 12 letters and must not
include full stop (.). The database is made from three separate
files. If the database is called FRED then version 0 of database
FRED comprises files FRED.AR0, FRED.RL0 and FRED.SQ0. The version is
the last symbol in the file names. Only this program can read these
files. If the "copy database" option is used it will ask the user to
define a new "version".
For normal use the maximum gel reading length is set to 512
characters, but when a database is started the user may choose
lengths of either 512, 1024, 1536..., 4096. Normally the program is
used to handle DNA sequences but many of the functions also work on
protein sequences. The choice of sequence type is made when the
database is started.
The contigs are not stored on the disk as the user sees them
displayed on the screen. Each gel reading is stored with sufficient
information about how it overlaps other gel readings so that the
program can work out how to present them aligned on the screen. We
refer to this extra data as "the relationships" and it is explained
below. The database comprises 3 separate files.
1. a working version of each gel reading. This is the version of
the gel reading that is in the database and initially it is an
exact copy of the original sequence (known as the archive) but it is
edited and manipulated to align it with other gel readings.
2. the file of relationships. This file contains all of the
information that is required to assemble the working versions into
contigs during processing; any manipulations on the data use this
file and it is automatically updated at any time that the
relationships are changed. The information in this file is as
follows:
(A) Facts about each gel reading and its relationship to
others ("gel descriptor lines"):
(a) the number of the gel reading (each gel reading is given a
number as it is entered into the database)
(b) the length of the sequence from this gel reading
(c) the position of the left end of this gel reading relative to
the left end of the contig of which it is a member
(d) the number of the next gel reading to the left of this gel
reading
(e) the number of the next gel reading to the right
(f) the relative strandedness of this gel reading , ie whether it
is in the same sense or the complementary sense as its archive.
(B) Facts about each contig ("contig descriptor lines"):
(a) the length of this contig
(b) the number of the leftmost gel reading of this contig
(c) the number of the rightmost gel reading of this contig.
(C) General facts:
(a) the number of gel readings in the database
(b) the number of contigs in the database.
3. the file of archive names. This is simply a list of the names
of each of the archive files in the database but on line number 1000
we also store the size of the database. ie the number of lines of
information allowed in the database files. This file always has 1000
lines but the length of the file of relationships and the file of
working versions can be set by the user when creating a database or
when copying from one to another.
Structure of the database files
1. The file of relationships
The file contains IDBSIZ lines of data: the general data are
stored on line IDBSIZ; data about gel readings are stored from
line 1 downwards; data about contigs are stored from line IDBSIZ-1
upwards. A database of 500 lines containing 25 gel readings and 4
contigs would have a file of relationships as is shown below.
---------------------------------------------
1 Gel descriptor record
2 " " "
3 " " "
4 " " "
5 " " "
' ' ' '
' ' ' '
25 " " "
26 Empty record
' ' '
' ' '
495 ' '
496 Contig descriptor record
497 " " "
498 " " "
499 " " "
500 Number of gel readings=25, Number of contigs=4
---------------------------------------------
The arrangement of the data in the file of relationships
As each new gel reading is added into the database a new line is
added to the end of the list of gel descriptor lines. If this
new gel reading does not overlap with any gel readings already in
the database a new contig line is added to the top of the list
of contig lines. If it overlaps with one contig then no new contig
line need be added but if it overlaps with two contigs then
these two contigs must be joined and the number of contig lines
will be reduced by one. Then the list of contig lines is compressed
to leave the empty line at the top of the list. Initially the two
types of line will move towards one another but eventually, as
contigs are joined, the contig descriptor lines will move in the
same direction as the gel descriptor lines. At the end of a
project there should be only one contig line. The database is
thus capable of handling a project of 998 gels.
Structure of the working versions file
The working versions of gel readings are stored in a file
of IDBSIZ lines each containing 512 characters. Gel reading number
1 is stored on line 1, gel reading number 2 on line 2 and so on.
Structure of the archive names file
This file, unlike the others, always has 1000 lines each 10
characters in length. Its length is fixed because line 1000 is used
to store IDBSIZ the database size and the programs need a definite
location from which to read this number.
Safeguarding the database
It is advisable to copy regularly (using the copy function of
DS) from say copy 0 to copy 1 in case of errors.
I also recommend setting the protection codes on copy 0 of
each database so that users cannot delete the files without first
resetting the protection codes. This will protect you from
accidently deleting the files. Users at LMB can use the PROTECT
command for this purpose.
The give-up options allow users to change their minds about
entering a new gel reading or joining two contigs without
affecting the file of relationships. BUT if the edit contig
option from either of these two functions has been used the
edits will remain even though the user has "given up". To leave the
files completely unaffected the user could, if required, undo
any edits before "giving up".
There are various checks within the programs to protect
users from themselves:-
1. All user input is checked for errors - e.g. reference to
non-existent gel readings or contigs, incorrect positions in the
contig or gel readings.
2. Before entering a gel reading the system checks to see if a file
of the same name has already been entered.
3. Join will not allow the circularising of a contig.
4. Both enter and join functions restrict the region that
the user is allowed to edit (using edit contig) to the region of
overlap.
5. Users may escape from any point in the program.
6. Help is available from all points in the program.
IT IS ESSENTIAL THAT USERS DO NOT KILL THE PROGRAM WHILE IT IS DOING
ANYTHING THAT INVOLVES CHANGING THE CONTENTS OF THE DATABASE. I.E
DURING AUTO ASSEMBLE, COMPLETE ENTRY, COMPLETE JOIN, COMPLEMENT
CONTIG, EDIT CONTIG, AND SCREEN EDIT. This could corrupt the
database so badly that it is impossible to fix. The program should
always be left using the QUIT option.
@4. TX 3 @Edit
A simple commnd driven editor that can insert, delete and
change gel reading sequences. Insert, delete and change commands
will request the position at which the edit is required and the
number of characters to insert, delete or change. The default
character for insertions is *.
There are three modes of editing offered by this editor
depending where it is selected from. New gel readings can be edited
as they are being entered into the database, contigs can be edited
with alignments being automatically maintained, or gel readings in
contigs can be edited without the maintenance of alignments.
The following commands can be used.
? = Help
! = Quit
3 = Insert
4 = Delete
5 = Change
All commands request the position at which the edit should be
made. (Note that the position refers to the position in the contig
for gel readings in the database, but to the position in the gel
reading if you are editing a new gel reading while entering it into
the database.)
All commands request the number of characters to operate on.
(Note that if you are editing a contig the program will ask for the
characters to insert into each separate gel reading, hence allowing
different changes to be made to each. Also the default character is
asterisk (*) - i.e if you include a space in the string it will be
replaced by an asterisk, or if you simply type return the whole
string inserted will be asterisks.)
"Change" allows characters in individual gel readings to be
replaced. If the user is not editing a new gel reading during
"enter new gel reading" the program will request the numer of the
gel reading to edit. (When editing gel readings in contigs the
program responds with the relative position and length of the
selected gel reading in case the the user only knows the edit
position relative to the gel reading. (The edit position must
be relative to the contig.))
Further notes on editing
When you are editing a contig the program maintains the
alignments of the gel readings by always making the same number of
insertions or deletions in all the gels. Note that these edits are
immediately carried out and the "Quit" options of "enter new gel
reading" and "join contigs" do not undo them. Users must undo them
themselves. Note that if this option has been entered from either
"enter new gel reading" or "join contigs" the program will restrict
edits to the region of overlap. DO NOT KILL THE PROGRAM DURING
EDIT CONTIG!
When editing a single gel reading in a contig from "alter
relationships" (which you should not normally need to do) the
program will correct the length of the individual gel reading, but
it will not update the length of the contig if it has changed.
The program contains better methods than this simple command
driven editor, for making multiple edits to contigs. "Screen edit",
gives access to the system editor on your machine, and "auto edit"
will edit a whole contig automatically.
@9. TX 3 @Screen edit
Gives access to the system editor on the machine (for example
EDT on a VAX) and allows users to edit contigs. The contigs are
presented as for "display contig" and the program will reconstitute
the contig's sequences and relationships when the editor is exited.
To screen edit a contig set the line length to 50 characters,
select the contig to edit, and supply the name of a temporary file
in which the editing will be performed. After a short pause the
system editor will present the first page of the file. Edit the file
obeying the rules given below. Exit from the editor and affirm the
intention of returning the contig to the database. The program will
put the contig back into the database.
Rules for screen editing
There are some limitations on the changes that can be made to
the contigs when using the screen editor. Users are unlikely to want
to break the rules in order to achieve changes to contigs, but
nevertheless the constraints need to be defined and they are given
below.
Alignments must be maintained during editing. Whole lines of
sequence should not be deleted or added unless the order of the gel
readings in the contig is preserved. Each line in the contig
display consists of gel reading numbers, their names and 50
character sections of sequence. Insertions are limited in the
following way. No line of sequence can be extended rightwards more
than 10 characters beyond the end of a full length line (a full
length line is 50 characters long). Only one character can be added
to the left end of full length lines, but sections of sequence
beginning further into a line can be extended leftwards up to an
equivalent position. Do not delete any non-sequence lines in the
file.
Before returning the contig to the database the program checks
that the rules have been obeyed. If an error is found the number of
the erroneous line in the file is displayed and the contig will not
be changed.
@5. TX 1 @Display a contig
Used to show the aligned gel readings for any part of a
contig. The number, name and strandedness of each gel reading is
shown and the consensus is written below.
If required identify the contig, and then the start and end
points of the region to display.
The display can be directed to a disk file using "direct
output to disk". These files are required by options: "screen edit"
and "highlight disagreements", and printed copies of them are very
useful for marking corrections prior to using the editors.
Below is an example showing the left end of a contig from
position 1 to 200. Overlapping this region are gels 6,3,5,17and
12; 6, 3 and 5 are in reverse orientation to their archives (denoted
by a minus sign) There are a few uncertainty codes and a few
padding characters in the working versions, but the consensus
(shown below each page width) has a definite assignment for almost
every position.
10 20 30 40 50
-6 HINW.010 GCGACGGTCTCGGCACAAAGCCGCTGCGGCGCACCTACCCTTCTCTTATA
CONSENSUS GCGACGGTCTCGGCACAAAGCCGCTGCGGCGCACCTACCCTTCTCTTATA
60 70 80 90 100
-6 HINW.010 CACAAGCGAGCGAGTGGGGCACGGTGACGTGGTCACGCCGCGGACACGTC
-3 HINW.007 GGCACA*GTC
CONSENSUS CACAAGCGAGCGAGTGGGGCACGGTGACGTGGTCACGCCG-G-ACA-GTC
110 120 130 140 150
-6 HINW.010 GATTAGGAGACGAACTGGGGCG3CGCC*GCTGCTGTGGCAGCGACCGTCG
-3 HINW.007 GATTAG4AGACGAACTGGGGCGACGCCCG*TGCTGTGGCAGCGACCGTCG
-5 HINW.009 GGCAGCGACCGTCG
17 HINW.999 AGCGACCGTCG
CONSENSUS GATTAGGAGACGAACTGGGGCGACGCC-G-TGCTGTGGCAGCGACCGTCG
160 170 180 190 200
-6 HINW.010 TCT*GAGCAGTGTGGGCGCTG*CCGGGCTCGGAGGGCATGAAGTAGAGC*
-3 HINW.007 TCT*GAGCAGTGTGGGCGCTGC*CGGGCTCGGAGGGCATGAAGTAGAGC*
-5 HINW.009 TCT*GAGCAGTGTGGGCG*T*G*CGGGCTCGGAGGGCATGAAGTAGAGC*
17 HINW.999 TCTCGAGCAGTGTGGGCGCTG**CGGGCTCGGAGGGCATGAAGTAGAGCG
12 HINW.017 GTAGAGC*
CONSENSUS TCT*GAGCAGTGTGGGCGCTG-*CGGGCTCGGAGGGCATGAAGTAGAGC*
@6. TX 1 @List a text file
This option allows users to list text files on the screen. It
can be used to read a file containing notes, for checking files
written to disk etc. The user is asked to type the name of the file
to list.
@8. TX 1 @Calculate a consensus
Calculates a consensus sequence either for the whole
database or for selected contigs. The consensus is written to a file
named by the user.
Supply a file name, choose between whole database or selected
contigs.
Symbols for uncertainty in gel readings
In order to record uncertainties when reading gels the
codes shown below can be used. Use of these codes permits us to
extract the maximum amount of data from each gel and yet record any
doubts by choice of code. The program can deal with all of
these codes and any other characters in a sequence are treated
as dash (-) characters.
SYMBOL MEANING
1 PROBABLY C
2 " T
3 " A
4 " G
D " C POSSIBLY CC
V " T " TT
B " A " AA
H " G " GG
K " C " C-
L " T " T-
M " A " A-
N " G " G-
R A OR G
Y C OR T
5 A OR C
6 G OR T
7 A OR T
8 G OR C
- A OR G OR C OR T
a A set by auto edit
c C set by auto edit
g G set by auto edit
t T set by auto edit
* padding character placed by auto assembler
else = -
The DNA consensus algorithm
The "calculate consensus" function, the "display contig"
routine and the "show quality" option use the rules outlined here
to calculate a consensus from aligned gel readings. Note that
"display contig" calculates a consensus for each page width it
displays (it does not use the consensus sequence file calculated
by the consensus function).
We have 6 possble symbols in the consensus sequence: A,C,G,T,*
and -. The last symbols is assigned if none of the others makes up a
sufficient proportion of the aligned characters at any position in
the contig. The following calculation is used to decide which symbol
to place in the consensus at each position.
Each uncertainty code contributes a score to one of A,C,G,T,*
and also to the total at each point. Symbols like R and Y which
don't correspond to a single base type contribute only to the total
at each point. The scores are shown below.
definite assignments ie A,C,G,T,B,D,H,V,K,L,M,N,a,c,g,t,* =1
probable assignments ie 1,2,3,4 = 0.75
other uncertainty codes including R,Y,5,6,7,8,- = 0.1
A cutoff score of 51% to 100% is supplied by the user. (When
the program starts this is set to 75%. See "set display
parameters"). At each position in the contig we calculate the total
score for each of the 5 symbols A,C,G,T and * (denote these by Xi,
where i=A,C,G,T or *), and also the sum of these totals (denote this
by S). Then if 100 Xi / S > the cutoff for any i, symbol i is placed
in the consensus; otherwise - is assigned.
Notice that S does not equal the number of times the sequence
has been determined, but is the score total, and hence we are less
likely to put a - in the consensus. For the "examine quality"
algorithm each strand is treated separately but the calculation is
the same. (It was originally different).
Format of the consensus sequence ( and vector sequences).
A consensus sequence file may contain the consensus for
several contigs and so we identify each of them by preceding them by
a 20 character title. The title is of the form <---LAMBDA.076----->
( where LAMBDA is the project name and gel reading number 76 is the
leftmost gel reading to contribute to this consensus sequence).
The angle brackets <> and the three digit number precede by a .
are important to some processing programs.
@25. TX 1 @Show relationships
Used to show the relationships of the gel readings in the
database in three ways -
(a) All contig descriptor lines followed by all gel descriptor
lines.
(b) All contigs one after the other sorted, i.e. for each
contig show its contig descriptor line followed by all its gel
descriptor lines sorted on position from left to right
(c) Selected contigs: show the contig line and, in order, those
gel readings that cover a user-defined region. Note that this
output can be directed to a disk file by prior selection of "disk
output".
Below is an example showing a contig from position 1 to 689.
The left gel reading is number 6 and has archive name HINW.010, the
rightmost gel reading is number 2 and is has archive name HINW.004.
On each gel descriptor line is shown: the name of the archive
version, the gel number, the position of the left end of the gel
reading relative to the left end of the contig, the length of
the gel reading (if this is negative it means that the gel reading
is in the opposite orientation to its archive), the number of the
gel reading to the left and the number of the gel reading to the
right.
CONTIG LINES
CONTIG LINE LENGTH ENDS
LEFT RIGHT
48 689 6 2
GEL LINES
NAME NUMBER POSITION LENGTH NEIGHBOURS
LEFT RIGHT
HINW.010 6 1 -279 0 3
HINW.007 3 91 -265 6 5
HINW.009 5 137 -299 3 17
HINW.999 17 140 273 5 12
HINW.017 12 193 265 17 18
HINW.031 18 385 -245 12 2
HINW.004 2 401 -289 18 0
@21. TX 3 @Enter new gel reading
Used to enter new gel readings into the database. The new gel
reading must have previously been compared with the contents of the
database by use of " auto assemble" in order to ascertain if it
overlaps any previously entered data.
The user is expected to know: if the gel reading overlaps; if
so which contig it overlaps; if so where it overlaps. The program
takes the user through a series of question to establish the nature
of the overlap and then displays the overlap. The user is then
offered a number of options, including editors for the new gel
reading and the contig, to enable the correct alignment of the gel
reading throughout its whole length.
Supply the name of the gel reading file. If the gel reading has
been entered before the program will not permit entry. The program
gives the gel reading a unique number and asks if the sequence
overlaps any data already in the database (reported by "auto
assemble"). If it does not, entry is complete. If it does overlap
the dialogue continues with the program asking if the gel readings
overlaps "in the normal sense", if not it will automatically
complement the sequence. Then supply the number of the contig the
gel reading overlaps (as reported by "auto assemble").
Overlaps are divided into two types: those for which the new
gel reading protrudes from the left end of the contig it overlaps,
and those for which it does not. The program asks about this with
the question "Left end of gel reading is inside contig". If this is
true the program will go on to ask for the position in the contig of
the left end of the new gel reading. If it is not true the program
will ask for the position in the new gel reading of the left end of
the contig.
Once this is completed the program will display the first 50
bases of the overlap. The gel readings in the contig and their
consensus are displayed with the new gel reading underneath. The
mismatches are shown by *'s on the next line down. For example:
60 70 80 90 100
-6 HINW.010 CACAAGCGAGCGAGTGGGGCACGGTGACGTGGTCACGCCGCGGACACGTC
-3 HINW.007 GGCACA*GTC
CONSENSUS CACAAGCGAGCGAGTGGGGCACGGTGACGTGGTCACGCCG-G-ACACGTC
NEWGEL CACAAGCGAGCGAGAGGGGCACCGTGACGTGGTCACGCCGGGGACACGTC
MISMATCH * * *
10 20 30 40 50
The program then needs to know if the position of the left
end of the overlap is correct. If it is the user should type
return, if not, 1 and the program will ask for the new position and
display it.
The program now offers a number of options to allow the user to
align the new gel reading correctly over its whole length with the
data already in the contig. It is important that
sufficient edits are made to the new gel reading or the
sequences in the contig at this stage to get the alignment correct,
because once entry is completed, the alignment is fixed and cannot
easily be changed (see "alter relationships"). Alignment can be
achieved by making insertions or deletions but deletion of
data requires the original gels to be checked. For this reason
at entry we usually make only insertions to achieve alignment. We
use X or asterisks (*) as padding characters to achieve alignment
and so can, if required, distinguish padding characters from
characters assigned from reading gels.
The options available are:
? = HELP
! = Give up
3 = Complete entry
4 = Edit contig
5 = Display overlap
6 = Edit new gel reading
1. HELP gives this information.
2. Give up allows users to change their minds about entering
the new gel reading. The program will ask the user to confirm this
choice.
3. Complete entry is the command to add the new gel reading to
the contig. The program updates the relationships accordingly. The
user is asked to confirm this command.
4. Edit contig gives the user access to a simple editor that
allows insertions, deletions and changes to be made to the contig.
The editor maintains alignments by making the same number of
insertions or deletions in all sequences covering the edit position.
The program protects the user by allowing edits only
within the region of overlap.
5. Display allows display of the region of overlap only. This
is defined by the relative positions in the contig. The default is
the whole of the region of overlap.
6. Edit new gel reading allows the new gel reading to be
edited using a simple editor.
@23. TX 3 @Complement a contig
This function will complement and reverse all of the gel
readings in a contig. It automatically reverses and
complements each gel reading sequence, reorders left and right
neighbours, recalculates relative positions and changes each
strandedness.
The only user input required is to identify the contig
to complement by the number or name of a gel reading it contains.
DO NOT KILL THE PROGRAM DURING THIS STEP!
@22. TX 3 @Join contigs
This function joins contigs interactively. It allows the user
to align the ends of the two contigs by editing each contig
separately. It is important that the alignment achieved is
correct because once the join is completed the alignment is fixed.
The program needs to know which two contigs to join and where they
overlap.
First which two contigs are to be joined. The user should
identify the two contigs. First the left contig and then the right.
The program checks that the two contig numbers are different (it
will not allow circles to be formed!)
Now identify the exact position of overlap. This is defined as
the position in the left contig that the leftmost character of the
right contig overlaps. Normally the position is established by
employing the end gel reading for option "auto assemble". The
overlap must be of at least one character. The program then
displays the join showing all the gel readings overlapping the
join from the left contig, their consensus, all the gel readings
from the right contig that overlap the join, their consensus
and then asterisks to denote mismatches between the two
consensuses. For example:
1460 1470 1480 1490 1500
56 HINW.100 TCT*GAGCAGTGTGGGCGCTG*CCGG
33 HINW.300 TCT*GAGCAGTGTGGGCGCTGC*CGGGCTCGGAGGG
-25 HINW.090 TCT*GAGCAGTGTGGGCG*T*G*CGGGCTCGGAGGG
19 HINW.123 TCTCGAGCAGTGTGGGCGCTG**CGGGCTCGGAGGGCATGAAGTAGAGCG
CONSENSUS TCTCGAGCAGTGTGGGCGCTG-CCGGGCTCGGAGGGCATGAAGTAGAGCG
-6 HINW.010 TCTCGAGCAGTGTGGGCGCTGCCCGGGCTCGGAGGGCATGAAGTTAGAGC
-3 HINW.007 TGGGCGCTGCCCGGGCTCGGAGGGCATGAAGT*AGAGC
-5 HINW.009 GCTCGGAGGGCATGAAGT*AGAGC
CONSENSUS TCTCGAGCAGTGTGGGCGCTGCCCGGGCTCGGAGGGCATGAAGTTAGAGC
MISMATCH * ******
10 20 30 40 50
It is essential that the user aligns the two contigs
throughout the whole region of overlap before completing the join
because it is only at this stage that the two contigs can be edited
independently. Once the join is completed the alignment can only be
altered using the routines supplied by "alter relationships". The
program offers the user options to facilitate the alignment of the
two contigs. These options are:-
? = Help
! = Give up
3 = Complete join
4 = Edit left contig
5 = Display joint
6 = Edit right contig
7 = Move join
1. Help gives this information.
2. Give up allows the user to return to the main options without
completing the join. Note any edits made will remain.
3. Complete join instructs the program to update the relationships
so that the two contigs are joined. DO NOT KILL THE PROGRAM DURING
COMPLETE JOIN!
4. Edit left contig and edit right contig give access to a simple
editor that allows insertions, deletions and changes to be made to
the contigs. Help is available on editing once the editing option
is selected. The user is only allowed to edit within the region of
overlap and should make sure that the positions used correspond to
the correct contig.
5. Display join displays the joint as shown above.
6. See above.
7. Move join allows the position of the joint to be changed.
@24. TX 1 @ Copy the database
Used to make a copy of the database. If required the database
size can be altered using this option. The "version" of a database
is encoded as the last letter in the names of the three files that
contain the database.
Supply a "version" number (the default is version 1), and if
required select a new size for the database. The size of a database
is the number of lines of information it can hold. It needs a line
for each gel reading and another for each contig.
@19. TX 1 @ Check database
Used to perform a check on the logical consistency of the
database. No user intervention is required.
The following relationships are checked:
1. If gel reading A thinks gel reading B is its left neighbour
does B think A is its right neighbour? The error message is
"Hand holding problem for gel reading A"
followed by the gel descriptor lines for gel readings A and B.
2. Are there any contig lines with no left or right end gel
readings? The error message is
"Bad contig line number A"
3. Do the gel readings that are described as left ends on
contig lines agree that they are left ends? The error message is
"The end gel readings of contig A have outward neighbours"
4. Are there gel readings that are in more than one contig?
The error message is
" Gel number A is used N times"
5. Are there gel readings that are not in any contig? The
error message is
" Gel number A is not used"
6. Do the relative positions of gel readings agree with
their position as defined by left and right neighbourliness? The
error message is
" Gel number A with position X is left neighbour of gel number B
with position Y"
7. Are there any loops in contigs? If so no further
checking is done. The error mesage is
" Loop in contig n no further checking done, but gel reading numbers
follow"
The program then prints the gel reading numbers in the looped
contig up to the start of the loop.
8. Are there any contigs of length <1? The error message is
" The contig on line number x has zero length"
9. Are there any gel readings (used in only one contig) that have
zero length? The error message is
" Gel number N has zero length"
Note that "auto assemble" also uses this logical consistency check
and will only tolerate a "Gel number N is not used" error. Any other
error will cause it to give up.
@29. TX 1 @ Examine quality
Analyses the quality of the data in a contig. It reports on
the proportion of the consensus that is "well determined" and will
display a sequence of symbols that indicate the quality of the
consensus at each position.
Identify the contig to analyse, and the section of interest.
The current consensus calculation cutoff score will be used to
decide if each position is "well determined". In general the quality
of a reading deteriorates along the length of the gel and so it is
also possible to use a length cutoff for the quality calculation.
Only the data from the first section of each reading will be
included in the quality calcualtion. The length is altered under
"set parameters" and is initially set to the maximum reading length.
A summary showing the percentage of the consensus that falls into
each category of quality is shown. Choose whether or not to have the
quality codes for each position of the consensus displayed. They can
be displayed as either graphics or text.
The quality of the data depends on the number of times it has
been sequenced and the particular uncertainty codes used in each
gel reading. This function divides the data into five categories,
assigning each a symbol or code:
1. Well determined on both strands and they agree. code=0
2. Well determined on the plus strand only. code=1
3. Well determined on the minus strand only. code=2
4. Not well determined on either strand. code=3
5. Well determined on both strands but they disagree. code=4
A position is "well determined" if it is assigned one of the symbols
A,C,G,T when the algorithm described in the section "calculate a
consensus". The calculation is performed separately for each
strand.
If the user chooses to have the data displayed graphically the
following scheme is used. A rectangular box is drawn so that the x
coordinate represents the length of the contig. The box is
notionally divided vertically into 5 possible levels which are given
the y values: -2,-1,0,1,2. The quality codes attributed to each
base position are plotted as rectangles. Each rectangle represents
a region in which the quality codes are identical, so a single base
having a different code from its immediate neighbours will appear as
a very narrow rectangle.
Rectangle bottom and top y values
Quality 0 rectangle from 0 to 0
Quality 1 rectangle from 0 to 1
Quality 2 rectangle from 0 to -1
Quality 3 rectangle from -1 to 1
Quality 4 rectangle from -2 to 2
Obviously a single line at the midheight shows a perfect
sequence.
Typical dialogue is shown below.
41.47% OK on both strands and they agree(0)
55.48% OK on plus strand only(1)
2.08% OK on minus strand only(2)
0.97% Bad on both strands(3)
0.00% OK on both strands but they disagree(4)
? (y/n) (y) Show sequence of codes
10 20 30 40 50
1111111111 1111111111 1111111111 1111111111 1111111111
60 70 80 90 100
1111111111 1111111111 1111111111 3111111111 1111111111
110 120 130 140 150
1111111111 1111131111 1111111111 1111111111 1111111111
160 170 180 190 200
1111111111 1111111111 1111111111 1111111111 1111111133
210 220 230 240 250
1311111111 1111111111 1111111110 0000000000 0000220000
260 270 280 290 300
0000000000 0020000000 2200000202 0002000000 0000222200
@26. TX 3 @ Alter relationships
Used to make what are normally illegal changes to the
database. That is the normal checks are not done and any item in the
database can be changed independently of all others. Users need to
know what they are doing because it is very easy to make a horrible
mess. Always start by making a copy!
By using the options here users can edit individual gel
readings in contigs, move one section of a contig relative to
another, break contigs, remove contigs, remove gel readings, etc. To
give flexibility most of the commands do only one thing. This means
that several commands may have to be executed to complete any
change. At the end of this help section there are notes on removing
gel readings from the database.
The following options are offered:
? = HELP
! = QUIT
3 = Line change
4 = Edit single gel reading
5 = Delete contig
6 = Shift
7 = Move gel reading
8 = Rename gel reading
9 = Break a contig
1. HELP gives this information.
2. QUIT returns to the main options of SAP.
3. Line change
allows the user to change the contents of any line in the file of
relationships. The line is selected by number, the program prints
the current line and prompts for the new line.
4. Edit
allows the user to edit an individual gel reading
independently of any others it may be related to. The edit positions
are relative to the contig. The effect of this editing on the length
of the gel reading is taken care of but, if it changes the length of
a contig, or its relationship to others, this must be accounted for
(if necessary) by use of the "line change" function.
5. Delete contig
is a function that deletes a contig line by moving down all the
contig lines above by one position. It prompts only for the line to
delete. It does not delete any of the gel readings or gel
reading lines for the deleted contig but it does reduce the number
of contigs on line IDBSIZ by 1.
6. Shift
allows the user to change all the relative positions of a set of
neighbouring gel readings by some fixed value, i.e. it will shift
related gel readings either left or right. It can therefore be
used to change the alignment of the gel readings in a contig or as
part of the process of breaking a contig into two parts (see below).
It prompts for the number of the first gel reading to shift and
then for the distance to move them (Note a negative value will
move the gel readings left and a positive value right). It then
chains rightwards (ie follows right neighbours) and shifts each gel
reading, in turn, up to the end of the contig. (This means that
only those gel readings from the first to shift to the rightmost are
moved). It updates the length of the contig accordingly.
7. Move gel reading
is a function to renumber a gel reading. It moves all the
information about a gel reading on to another line. The user must
specify the number of the gel reading to move and the number of the
line to place it. It takes care of all the relationships. Of course
gel readings must not be moved to lines occupied by other gel
readings! It can be used as part of the process of removing a gel
reading from the database (see below).
8. Rename gel reading
is a function that is used to rename the archive names of gel
readings in the database; it only changes the name in the .ARN
file of the database.
9. Break contig
Occasionaly it is necessary to break a contig into two parts
and this can be achieved using this option. The program needs only
the number of a gel reading. This is the gel reading that will
become a left end after the break. That is, the break is made
between this gel reading and its left neighbour. A new contig line
is created so ensure that there is sufficient space in the database.
Removing gel readings from contigs
Gel readings can be removed from contigs if they are not
essential for holding the contig together (ie are not the only gel
reading covering a particular region). Suppose the gel reading to
remove is gel number b with left neighbour a and right neighbour c.
Using "line change" change the right neighbour of a to c, and the
left neighbour of c to a. To tidy things up: suppose there are x gel
readings in the database; then, using "move gel reading" move gel x
to line b; then, using "line change" decrease the number of gel
readings in the database (stored in the last line) by 1.
@27. TX 1 @ Set display parameters
Used to redefine the parameters that control the cutoff
employed by the consensus calculation and quality examiner, the
maximum length of each reading to include in the quality
calculation, the line length used by the display function, the text
window length used by the graphics options, and the graphics window
length used by the graphics options.
The default cutoff score is 75%. The default line length is 50
characters. For protein sequences the cutoff is always 100%.
The text window used by the graphics options controls the
amount of sequence listed at the crosshair position. The graphics
window controls the "zoom" function. Both these windows are defined
as the number of bases that should be shown, to both left and right
of the crosshair.
@30. TX 3 @ Auto edit a contig
This function automatically changes characters in gel readings
to make them agree with the consensus sequence. If employed as is
intended, use of this function is not a criminal activity but a
method that saves a large amount of work. All characters changed by
the auto editor will appear in the gel readings as lowercase
letters. The current consensus calculation cutoff score is used.
Identify the contig and the section to edit. The program will
display a summary of changes made. Note that it is important to
understand both what the auto editor does and the order in which it
does it. Before employing the auto editor users should note all the
corrections that they require, so that after it has been used the
corrections can be checked.
The general strategy employed when collecting shotgun sequence
data is to let the contigs get fairly deep, to get a printout of a
contig, check problems against the films, note corrections on the
printout, and make the changes using an interactive editor. In
general the consensus is correct except for places where padding
characters have been used to accommodate a single gel with an extra
character, or where the consensus is dash. The important point for
the auto editor is that most edits simply make the gel readings
conform to the consensus, or remove columns of pads.
The new editor does the following.
1) calculates a consensus for the contig (or part of a contig)
to be edited, and then uses this consensus to direct the editing of
the contig in 3 stages
2) stage 1: find and correct all places where, if the order of
two adjacent characters is swapped, they will both agree with the
consensus (given that they did not match the consensus before).
These corrections are termed "transpositions"
3) stage 2: find and correct all places where there is a
definite consensus but the gel reading has a different character.
These corrections are termed "changes".
4) stage 3: delete all positions in which padding is the
consensus. These corrections are termed "deletions".
All changed characters are shown in lowercase letters so it
will be obvious which characters have been assigned by the program
(except for deletions). The number of each type of correction will
be displayed.
@10. TX 2 @Clear graphics
Clears graphics from the screen.
@11. TX 2 @Clear text
Clears text from the screen.
@12. TX 2 @Draw a ruler.
This option allows the user to draw a ruler or scale along the
x axis of the screen to help identify the coordinates of points of
interest. The user can define the position of the first base to be
marked (for example if the active region is 1501 to 8000, the user
might wish to mark every 1000th base starting at either 1501 or 2000
- it depends if the user wishes to treat the active region as an
independent unit with its own numbering starting at its left edge,
or as part of the whole sequence). The user can also define the
separation of the ticks on the scale and their height. If required
the labelling routine can be used to add numbers to the ticks.
@14. TX 2 @Reposition plots
The positions of each of the plots is defined relative to a
users drawing board which has size 1-10,000 in x and 1-10,000 in y.
Plots for each option are drawn in a window defined by x0,y0 and
xlength,ylength. Where x0,y0 is the position of the bottom left hand
corner of the window, and xlength is the width of the window and
ylength the height of the window.
--------------------------------------------------------- 10,000
1 1
1 -------------------------------------- ^ 1
1 1 1 1 1
1 1 1 1 1
1 1 1 ylength 1
1 1 1 1 1
1 1 1 1 1
1 -------------------------------------- v 1
1 x0,y0^ 1
1 <---------------xlength--------------> 1
--------------------------------------------------------- 1
1 10,000
All values are in drawing board units (i.e. 1-10,000, 1-10,000).
The default window positions are read from a file "ANALMARG" when
the program is started. Users can have their own file if required.
As all the plots start at the same position in x and have the same
width, x0 and xlength are the same for all options. Generally users
will only want to change the start level of the window y0 and its
height ylength. This option allows users to change window positions
whilst running the program. The routine prompts first for the
number of the option that the users wishes to reposition; then for
the y start and height; then for the x start and length. Note that
changes to the x values affect all options. If the user types only
carriage return for any value it will remain unchanged. Note that,
unlike all the other programs, the boxes used to contain analytical
results (eg plot quality) should not be made to overlap one another,
as the function of the crosshair routine depends on which box the
crosshair is in! overlap
@15. TX 2 @Label a diagram
This routine allows users to label any diagrams they have
produced. They are asked to type in a label. When the user types
carriage return to finish typing the label the cross-hair appears on
the screen. The user can position it anywhere on the screen. If the
user types R (for right justify) the label will be written on the
diagram with its right end at the cross-hair position. If the user
types L (for left justify) the label will be written on the diagram
with its left end at the cross hair position. The cross-hair will
then immediately reappear. The user may put the same label on
another part of the diagram as before or if he hits the space bar he
will be asked if he wishes to type in another label.
Typical dialogue follows.
? Menu or option number=15
Type label then drive cross hair to left or right end
of label position then hit "L" to write label left
justified or "R" to write label right justified or
the space bar to quit
? Label=delta gene
missing graphics
? Label=
@16. TX 2 @Display a map.
This draws a map of any sequence features selected by the
user. These features may be protein coding regions (CDS), tRNA
genes (TRNA), promoter positions (PRM), etc. Users may define their
own feature table key names. For example I find it convenient to
split CDS lines into CDS1, CDS2 and CDS3 each of which contains only
those sequences that code in the reading frames 1, 2 or 3. Then I
can plot them at different heights on the screen ( suitable heights
can be determined by using the cross-hair). The coordinates must be
stored in a file in the format of an EMBL feature table.
Typical dialogue follows.
? Menu or option number=16
Display a map using an EMBL feature table file
? map file name=hsegl1.ft
? feature code(e.g. CDS) =CDS
X 1 + strand
2 - strand
3 both strands
? 0,1,2,3 =
? level (0-9480) (256) =4000
missing graphics
? feature code(e.g. CDS) =
@7. TX 1 @Redirect output
Used to direct output that would normally appear on the screen
to a file.
Select redirection of either text or graphics, and supply the
name of the file that the output should be written to.
The results from the next options selected will not appear on
the screen but will be written to the file. When option 7 is
selected again the file will be closed and output will again appear
on the screen.
@13. TX 2 @Use crosshair
This option puts a steerable cross on the screen which the user
drives around by using the arrow keys (or mouse). When the crosshair
is visible a number of options are available if the user types one
of a set of special keyboard characters. Any other characters will
cause an exit from the crosshair option. The special keys are:
I = Identify the nearest gel reading
Z = Zoom in
Q = plot Quality
S = display the aligned Sequences at the crosshair position
N = list the Names and Numbers of the sequences at the crosshair
In order for any of these special keys to operate, the
crosshair must be in an appropriate display box, and the precise
function of the keys will also depend on which box the crosshair is
in.
If the crosshair is in the "plot all contigs" box, Z will
cause a new box to appear showing all the readings for the nearest
contig; Q will give the same as Z but will also produce an extra box
showing the "quality" plot.
If Z is hit in the "plot single contig" box, the contig will
be zoomed to the current graphics window size. The zoom will be
roughly centred on the crosshair position. Because of this it is
possible to step along a contig by repeatedly zooming with the
crosshair near to one end of the single contig display box. If I is
hit the crosshair must be close to a gel reading line. If Q is hit,
the quality plot will be produced for the region shown in the plot
single contig box. In all cases when the "plot all contigs" box is
shown, a vertical line will bisect the line the represents the
relevent contig, at the current position.
If the crosshair is in the plot quality box only the character
"s" will operate as a special symbol.
The number of bases shown in the N and S options is controlled
by the current graphics text window size, and the size of the zoom
window by the current graphics window size. Both are set by the
parameter setting function of the general menu.
@33. TX 2 @Plot single contig
This option produces a schematic of a selected region of a single
contig by drawing a horizontal line to represent each of its gel
readings. The lines show the relative positions of each reading and
also their sense. The plot is divided vertically into two sections
by a line that is identified by an asterisk drawn at each end. All
lines that lie above this line represent readings that are in their
original sense, all lines below show readings that are in the
complementary sense to their original. By use of the crosshair
function the plot can be stepped through and examined in more
detail. See help on crosshair.
@34. TX 2 @Plot all contigs
This option produces a schematic of all the contigs in a database.
It does this by drawing a horizontal line to represent each of them.
In order to show the ends of each contig it draws the lines for
contigs at alternate heights: the first at height one, the second at
height two, the third at height one, etc. The order of the contigs
in the display is the same as their order in the database. By use of
the crosshair function the plot can be stepped through and examined
in more detail. See help on crosshair.
@31. TX 3 @ Type in gel readings
This option allows gel readings to be typed in at the keyboard. It
creates a separate file for each gel reading and a file of file
names for the batch. The sequences from each batch may be listed
when they have all been entered. Users may choose to employ special
keys to identify the 4 bases A,C,G and T. By default these special
keys are N M , . but any other four characters may be used. If
special keys are used the characters are automatically translated to
A C G T before being stored on the disk.
@35. TX 1 @ Find internal joins
The purpose of this function is to use data already in the database
to find possible joins between contigs. Joins may have been missed
due to poor data or may have not been made due to repeated
sequences. Where appropriate, it may be possible to find potential
joins by using the data clipped off readings prior to their entry
into the database.
The database is checked for logical consistency. Supply a minimum
initial match length, a minimum alignment block, the maximum pads
per sequence, the maximum percent mismatch after alignment, the
probe length. Choose if clipped data is to be used, if so define the
window size for finding good data and the number of dashes allowed
in the window. Processing will commence. Most of these values are
used in an identical way in the autoassemble function. The others
are defined below.
The program strategy
Take the first contig and calculate its consensus. If clipped data
is being used examine all readings that are in the complementary
orientation, and sufficiently near to the contigs left end, to see
if they have good clipped sequence which if present, would protrude
from the left end of the contig. If found add the longest such
sequence to the left end of the consensus. Do the same for the right
end by examining readings that are in their original orientation. If
any are found add the longest extension to the right end of the
consensus. Repeat the consensus calculations and extensions for all
contigs hence producing an extended consensus. If clipped data is
not being used simply calculate the consensus for the whole
database. Now look for possible joins by processing the extended
consensus in the following way. Take the last, say 100, bases
(termed the "probe length" by the program) of the rightmost
consensus, compare it both orientations with the extended consensus
of all the other contigs. Display any sufficiently good alignments.
Repeat with the left end of the rightmost contig. Do the same for
the ends of all the entended contigs, always only comparing with the
contigs to their left, so that the same matches do not appear twice.
Good cliped data is defined by sliding a window of "Window size for
good data scan" bases outwards along the sequence and stopping when
"Maximum number of dashes in scan window" or more dashes appear in
the window. Note that it is advisable to have some sort of cutoff
because if we simply take all the data it might be so full of
rubbish that we wont find any good matches. For the same reason it
is worth trying the procedure with different cutoffs. An initial run
using no clipped data is also recommended. Sufficiently good
alignments are defined by criteria equivalent to those used in
autoassemble, however here we only display alignments that pass all
tests.
Bugs
If a small contig is wholly contained within a larger one, such that
its ends are further than ("Probe length" - "Minimum initial match
length") from the ends of the larger contig, and the consensus for
the small contig lies to the left of the consensus for large contig,
the overlap will not be discovered. (See the search stratgey).
All numbering is relative to base number one in the contig: matches
to the left (i.e. in the clipped data) have negative positions,
matches off the right end of the contig (i.e. in the clipped data)
have positions greater than that of the contig length. A typical
result is shown below.
Right end of contig 22 in the - sense and contig 96
Percentage mismatch after alignment = 3.0
628 638 648 658 668 678
GTGAGATGAG CATATTTAAA ATGAACCGAG CAGTTAGGAG ATATGTTGGG AGGACAAGAA
********* ********** ********** ********** ********** **********
-TGAGATGAG CATATTTAAA ATGAACCGAG CAGTTAGGAG ATATGTTGGG AGGACAAGAA
-86 -76 -66 -56 -46 -36
688 698 708 718
ACATCCGGGA TACAGTCAAT AAATGAAAAA TTAATGAATT
********** ********** ****** *** ***** ****
ACATCCGGGA TACAGTCAAT AAATGA-AAA TTAATTAATT
-26 -16 -6 4
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