Molecular Biology Software

Training Manual

Molecular Biology Support
Scientific Applications Section
AFRC Computing Division
Harpenden
Herts AL5 2JE
UK

email: support@afrc.ac.uk

Tel:+44 582 762271 	Fax: +44 582 761710

May 1994

PREFACE
History
This document began, in 1989, as a set of exercises for a training course in the use of the GCG package. Its intention was to introduce research workers, including novice computer users, to molecular biology software. The current document now includes background notes and other software as part of the training course. The revised aim: to provide a brief introduction to the facilities available within the AFRC's VAX/VMS network called AGRENET, and beyond.

The document was never intended to be a comprehensive coverage of the subject. Many items of detail are omitted, which were explained during the course. If the user is prepared to experiment with data and explore on the basis of these examples, then this document may be of some use. Many users will find themselves using UNIX-based systems in which case the amendments for using GCG programs are quite minor: the command line options use a space and a minus instead of a slash key. eg: on page 3-6 use: mapsort -exclude=388,1020 -six/

Summary of contents
Chapter 1 is very AFRC-specific. Chapter 2 is a condensed GCG-reference section with some AFRC-specific information included. Chapters 3 to 16 contain worked exercises and background notes. All the examples show how the software behaves on Agrenet VAXes - some programs default to batch-submission, using local queue names. All sequences used in the exercises are obtainable from the exercises themselves, or can be located using Appendix B. Some of the worked examples may appear different on other VAX systems. A description of the local program amendments is given in Appendix B.

The course
The course presented was given over a period of two days, although a three day course might be more appropriate. The order of presentation in the manual is intended as starting with easier programs, moving to progressively more complex programs, or where greater explanation is required. A case is easily made for providing a course with the chapters in a completely different order.

Availability
This document is available on the EMBL file server (see Chapter 16.2.1) as MS-WORD files.

Acknowledgements
My thanks to the c.500 people who have attended the course for their comments, criticisms and suggestions for improvements. I am particularly grateful to the following for their advice and comments.

David Judge - Department of Genetics, University of Cambridge, U.K.
Sarah McQuay - BRU, Kings Buildings, University of Edinburgh, U.K.
Frank Wright - SASS, Kings Buildings, University of Edinburgh, U.K.

The May 1994 version of the document for MS-WORD was prepared at Glaxo Research & Development, UK; more thanks to Sarah McQuay.
	Cary O'Donnell 6th-Sep-1993, 20th May 1994

MOLECULAR BIOLOGY SOFTWARE

TRAINING MANUAL

CONTENTS
P
1. STARTING SEQUENCE ANALYSIS ON AGRENET	1-1
1.1.	Logging on	1-1
1.2.	Starting up the main packages	1-1
1.3.	Using LOGIN.COM	1-2
1.4.	Other software packages	1-2
1.5.	Help information	1-2
1.6.	Documentation	1-2
1.7.	Graphical output	1-2
1.7.1.	Unipict files	1-3
1.8.	Software and database availability on AGRENET	1-3
1.9.	Databases on AGRENET	1-4
1.9.1.	Nucleic acid databases	1-4
1.9.1.1.	Database divisions -	1-4
1.9.2.	Protein databases	1-4
1.9.3.	Brookhaven database	1-5
1.10.	Useful VMS commands	1-5
1.11.	Queues on AGRENET	1-5
1.11.1.	Batch queues	1-5
1.11.2.	Printer queues	1-6

2. THE GENETICS COMPUTER GROUP PACKAGE	2-1
2.1.	What is GCG ?	2-1
2.1.1.	Program examples	2-1
2.1.2.	Command line modifiers	2-1
2.2.	Databases with GCG	2-2
2.2.1.	Database sequence names	2-2
2.2.2.	Database accession numbers	2-2
2.2.3.	Searching databases	2-2
2.3.	Groups of sequences	2-3
2.3.1.	Files of sequence names	2-3
2.3.2.	Multiple sequence files	2-3
2.4.	Program default values	2-3
2.5.	Graphics with the GCG package	2-4
2.5.1.	Graphics driver-selection	2-4
2.5.2.	Fonts	2-4
2.6.	GCG figure files	2-4
2.7.	Local data files	2-5
2.7.1.	Enzyme data files	2-5
2.8.	Nucleotide symbols in GCG	2-6
2.9.	Amino acid symbols and the standard translation table	2-7
2.10.	Nucleotide symbol comparison table for BESTFIT	2-8
2.11.	PAM250 amino acid symbol comparison table	2-8
2.12.	BLOSUM62 amino acid symbol comparison table	2-9
2.12.1.	Analysis of symbol comparison values of  PAM250	2-10
2.13.	Programs in the GCG package (release 7.3)	2-12
2.13.1 Supplementary programs	2-15

3. GENERAL SEQUENCE MANIPULATION	3-1
3.1.	Help information and documentation	3-1
3.1.1.	GENHELP and EGENHELP - Help on GCG programs	3-1
3.1.2.	GENMANUAL - Help by program function	3-1
3.1.3.	EASYGCG - Finding your way	3-2
3.2.	Formatting a GCG sequence	3-2
3.2.1.	Formatting raw sequence data	3-2
3.2.2.	DNA Û RNA conversion	3-3
3.2.3.	Sequence complementing	3-3
3.3.	SEQHELP - Help for other analysis programs	3-4
3.4.	Copying a database sequence	3-4
3.5.	Restriction mapping programs	3-5
3.5.1.	MAP	3-5
3.5.1.1.	Selecting enzymes -	3-5
3.5.2.	MAPSORT (and selecting enzymes by region)	3-6
3.5.2.1.	Digest (and selecting enzymes by name) -	3-6
3.5.2.2.	Creating a plasmid map. -	3-7
3.5.3.	The enzyme list	3-7
3.5.4.	Protein sequence mapping	3-7
3.6.	Sequence editing	3-8
3.6.1.	Editing modes -	3-8
3.6.2.	Adding sequence data from a file -	3-8
3.6.3.	Moving around in the sequence -	3-8
3.6.4.	Editing comments -	3-9
3.6.5.	Writing part of the sequence to a file -	3-9
3.6.6.	Deleting part of the sequence -	3-9
3.6.7.	Help -	3-9
3.6.8.	Exiting -	3-9
3.6.9.	Changing your keyboard -	3-9
3.7.	Interconverting sequence formats	3-10
3.7.1.	Readseq	3-10
3.7.2.	PIR/NBRF format conversions	3-11
3.7.3.	STADEN format conversions	3-11

4. PROTEIN ANALYSIS	4-1
4.1.	Identifying open reading frames	4-1
4.2.	Identifying potential coding regions	4-2
4.2.1.	Base composition of bulk DNA	4-2
4.2.2.	Base composition in the third codon position	4-3
4.2.3.	Codon usage bias	4-3
4.3.	Translating RNA into protein	4-4
4.3.1.	Three and one-letter abbreviations	4-4
4.4.	Predicting secondary structure in proteins	4-5
4.4.1.	PEPTIDESTRUCTURE & PLOTSTRUCTURE	4-5
4.4.2.	MOMENT	4-6
4.4.3.	PEPPLOT	4-6
4.5.	Translating protein intoRNA	4-7
4.5.1.	Best sequence option	4-7
4.5.2.	Most ambiguous sequence option	4-7

5. COMPARING SEQUENCES	5-1
5.1.	Identifying sequence homology	5-1
5.1.1.	Word comparison	5-1
5.1.2.	Dotplotting	5-1
5.1.2.1.	Interpreting the plot -	5-1
5.1.2.2.	The effect of word size -	5-1
5.1.2.3.	Types of patterns -	5-2
5.1.3.	Window comparison	5-2
5.1.4.	Comparison of proteins	5-3
5.1.5.	Symbol comparison tables	5-3
5.2.	Sequence alignments	5-4
5.2.1.	BESTFIT	5-4
5.2.2.	GAP	5-4
5.2.3.	Protein sequence alignment	5-5
5.2.4.	Alignment measurements	5-5
5.2.4.1.	Quality	5-5
5.2.4.2.	Ratio	5-5
5.2.4.3.	Identity	5-5
5.2.4.4.	Similarity	5-5

6. SEARCHING DATABASES	6-1
6.1.	Searching by text - STRINGSEARCH	6-1
6.1.1.	Definition search	6-1
6.1.2.	Full text search	6-1
6.2.	Interactive text searches - XQS	6-2
6.2.1.	Nucleotide databases	6-2
6.2.2.	Protein databases	6-3
6.3.	Sequence homology search	6-4
6.3.1.	FASTA (direct search)	6-4
6.3.2.	TFASTA (translation search)	6-4
6.3.3.	Exhaustive homology searching	6-5
6.4.	Interpreting FASTA output	6-6
6.4.1.	The FASTA algorithm	6-6
6.4.1.1.	Disadvantages of the FASTA algorithm -	6-6
6.4.2.	A FASTA strategy	6-6
6.4.3.	Diagram of FASTA algorithm	6-7
6.4.4.	The histogram	6-8
6.4.5.	Mean scores and cpu time	6-8
6.4.6.	Example FASTA histogram:	6-9
6.4.7.	The best scores	6-10
6.4.8.	The alignments	6-11
6.5.	Interpreting PROSRCH output	6-12
6.5.1.	Data check	6-12
6.5.2.	Symbol comparison table	6-12
6.5.3.	Additional information	6-12
6.5.4.	How PROSRCH works	6-12
6.5.5.	Figure: score vs log (number of entries).	6-13
6.5.6.	The score distribution and statistics	6-14
6.5.7.	The alignments	6-15
6.5.8.	The individual alignment scores	6-15
6.5.9.	Score ratios and PAM tables	6-15
6.5.10.	Mapping	6-16
6.5.11.	Additional alignments	6-17
6.5.12.	A PROSRCH strategy	6-17
6.6.	Other Biosearch programs	6-17

7. MULTIPLE SEQUENCE ALIGNMENT	7-1
7.1.	Cluster alignments	7-1
7.1.1.	PILEUP	7-1
7.1.2.	GCLUSTALV	7-2
7.2.	Manual alignment	7-4
7.3.	Alignments displays	7-5
7.3.1.	Threshold, plurality and weightings	7-5
7.4.	Boxed graphic displays	7-6
7.4.1.	PRETTYPLOT	7-6
7.4.2.	PRETTYBOX	7-6

8. FRAGMENT ASSEMBLY SYSTEM	8-1
8.1.	Introduction	8-1
8.1.1.	Goals	8-1
8.1.2.	Summary of programs	8-1
8.2.	Fragment assembly tutorial	8-2
8.2.1.	NEWGELSTART	8-2
8.2.2.	GELENTER	8-2
8.2.3.	GELMERGE	8-3
8.2.4.	GELVIEW	8-3
8.2.5.	GELASSEMBLE	8-4
8.2.6.	GELOVERLAP	8-5
8.2.7.	GELENTER as an editor	8-6
8.2.8.	Redefining the project	8-6
8.2.9.	Detecting vector the unofficial way	8-6

9. FINDING SEQUENCE MOTIFS	9-1
9.1.	Locating partial sequences	9-1
9.2.	Protein motifs	9-2
9.2.1.	Retrieving PROSITE documentation	9-2

10. SEQUENCE PROFILING	10-1
10.1.	The profiling method	10-1
10.2.	Description of the profile table	10-1
10.2.1.	A profile as a symbol comparison table	10-2
10.3.	Finding a new member of the alignment	10-2
10.4.	Profiling tutorial	10-3
10.4.1.	PROFILEMAKE	10-3
10.4.2.	PROFILEGAP	10-3
10.4.3.	PROFILESEARCH	10-4
10.4.4.	PROFILESEGMENTS	10-4
10.4.5.	PROFILESCAN	10-5
10.5.	Nucleotide profiling	10-5

11. RNA SECONDARY STRUCTURE	11-1
11.1.	Identifying inverted repeats	11-1
11.2.	Calculating RNA folding	11-2
11.3.	Display of folding structures	11-2
11.4.	Alternative structures	11-3

12. GCG COMMAND FILES	12-1
12.1.	What are they?	12-1
12.2.	Editing GCLUSTALV.COM	12-1
12.3.	STRINGSEARCH	12-2
12.4.	Other command files	12-2

13. GCG DATA FILES	13-1
13.1.	Local data files	13-1
13.1.1.	Enzyme tables	13-1
13.1.2.	Codon usage (or codonpreference) tables	13-2
13.1.3.	Symbol comparison tables	13-2
13.1.4.	Translation tables	13-3
13.1.5.	Yet more data!!	13-3
13.2.	PLASMIDMAP files	13-4
13.2.1.	Displaying blocks and ranges	13-4

14. ORGANISING YOUR OWN DATABASES	14-1
14.1.	Using files of sequence names	14-1
14.2.	Create an indexed database	14-1

15. PHYLOGENY INFERENCING	15-1
15.1.	Starting Phylip	15-1
15.2.	MSF to Phylip	15-1
15.3.	The DNADIST program	15-1
15.4.	The NEIGHBOR and FITCH programs	15-2

16. BEYOND GCG...	16-1
16.1.	Submitting a sequence to the databases	16-1
16.1.1.	Copy the submission form	16-1
16.1.2.	Enter the details	16-1
16.1.3.	Mail the sequence	16-1
16.1.4.	Acknowledgement	16-1
16.1.5.	Authorin	16-1
16.2.	Obtaining software from remote sites	16-2
16.2.1.	The EMBL file server	16-2
16.2.2.	The Indiana ftpsite	16-2
16.3.	BIOSCI bulletin board	16-3
16.3.1.	Topics	16-3
16.3.2.	Subscription requests	16-4
16.3.3.	Sending a message to a bulletin board.	16-4
16.3.4.	Reading the bulletin board	16-4
16.3.5.	Cancelling subscriptions	16-5
16.3.6.	Biosci and local bulletins on Agrenet	16-5
16.4.	The Internet GOPHER	16-6
16.5.	MOSAIC- world tours by mouse	16-7

A. DATABASE SEARCH RESULTS	A-1
A.1	FASTA search of EMBL37 (Dec 1993) with a word size = 6	A-1
A.1.1	Histogram	A-1
A.1.2	The 100 best scores	A-2
A.1.3	The alignments	A-4
A.2	FASTA search of EMBL37 (Dec 1993) with a word size = 1	A-5
A.2.1	Histogram	A-5
A.2.2	The 100 best scores	A-6
A.3	TFASTA search of EMBL37 (Dec 1993) with a word size = 1	A-8
A.3.1	Histogram	A-8
A.3.2	The 100 best scores	A-9
A.3.3	A TFASTA alignment	A-11

B. MISCELLANEOUS	B-1
B.1.	Sequences used in the exercises	B-1
B.1.1	Main example mRNA sequence.	B-1
B.1.2	Other RNA sequences	B-1
B.1.3	Protein sequences	B-1
B.2.	Further reading	B-1
B.3.	Local program amendments	B-2
B.3.1	Program symbols	B-2
B.3.2	Batch queue command line option	B-2
B.3.3	Batch log files	B-2
B.3.3.1	CLSUBMIT.FOR differences listing:	B-3
B.3.4	GCLUSTALV	B-5
B.3.5	STRINGSEARCH and PROFILESEARCH	B-5
B.4.	Obtaining this document from the EMBL file server	B-6
B.5.	WORD for Windows source files	B-6

C. DOTPLOT DIAGRAMS	C-1
C.1	A sequence compared to itself	C-1
C.2	Sequence divergence	C-2
C.3	Deletions and insertions	C-2
C.4	Tandem duplication	C-3
C.5	Internal repeats	C-3

D. USING A SEQUENCE DIGITISER	D-1
D.1.	DIGISEQ	D-1
D.2.	Interpretation of sequence gels	D-2
D.2.1.	Band distribution	D-2
D.2.2.	Variable band intensity	D-2
1	D.2.2.1The C rules: -	D-2
2	D.2.2.2.The A rules: -	D-2
3	D.2.2.3.Other rules: -	D-2
D.3.	KERMIT file transfers	D-3
D.4.	EMUTEK file transfers	D-4