![]() |
merger |
It uses a global alignment algorithm (Needleman & Wunsch) to optimally align the sequences and then it creates the merged sequence from the alignment. When there is a mismatch in the alignment between the two sequences, the correct base to include in the resulting sequence is chosen by using the base from the sequence which has the best local sequence quality score. The following heuristic is used to find the sequence quality score:
If one of the bases is a 'N', then the other sequence's base is used, else:
A window size around the disputed base is used to find the local quality score. This window size is increased from 5, to 10 to 20 bases or until there is a clear decision on the best choice. If there is no best choice after using a window of 20, then the base in the first sequence is used.
To calculate the quality of a window of a sequence around a base:
N.B. This heavily discriminates against the iffy bits at the end of sequence reads.
This program was originally written to aid in the reconstruction of mRNA sequences which had been sequenced from both ends as a 5' and 3' EST (cDNA). eg. joining two reads produced by primer walking sequencing.
Care should be taken to reverse one of the sequences (e.g. using the qualifier '-sreverse2') if this is required to get them both in the correct orientation.
Because it uses a Needleman & Wunsch alignment the required memory may be greater than the available memory when attempting to merge large (cosmid-sized or greater) sequences.
The gap open and gap extension penalties have been set at a higher level than is usual (50 and 5). This was experimentally determined to give the best results with a set of poor quality EST test sequences.
% merger Merge two overlapping nucleic acid sequences Input sequence: tembl:eclacy Second sequence: tembl:eclaca Output sequence [eclacy.fasta]: Output alignment [eclacy.out2]:
Typically, one of the sequences will need to be reverse-complemented to put it into the correct orientation to make it join. For example:
% merger file1.seq file2.seq -sreverse2 -outseq merged.seq -outfile stdout
Mandatory qualifiers: [-seqa] sequence Sequence USA [-seqb] sequence Sequence USA [-outseq] seqout Output sequence USA [-outfile] align Output alignment and explanation Optional qualifiers: -datafile matrixf This is the scoring matrix file used when comparing sequences. By default it is the file 'EBLOSUM62' (for proteins) or the file 'EDNAFULL' (for nucleic sequences). These files are found in the 'data' directory of the EMBOSS installation. -gapopen float Gap opening penalty -gapextend float Gap extension penalty Advanced qualifiers: (none) General qualifiers: -help boolean Report command line options. More information on associated and general qualifiers can be found with -help -verbose |
Mandatory qualifiers | Allowed values | Default | |
---|---|---|---|
[-seqa] (Parameter 1) |
Sequence USA | Readable sequence | Required |
[-seqb] (Parameter 2) |
Sequence USA | Readable sequence | Required |
[-outseq] (Parameter 3) |
Output sequence USA | Writeable sequence | <sequence>.format |
[-outfile] (Parameter 4) |
Output alignment and explanation | Alignment file | |
Optional qualifiers | Allowed values | Default | |
-datafile | This is the scoring matrix file used when comparing sequences. By default it is the file 'EBLOSUM62' (for proteins) or the file 'EDNAFULL' (for nucleic sequences). These files are found in the 'data' directory of the EMBOSS installation. | Comparison matrix file in EMBOSS data path | EBLOSUM62 for protein EDNAFULL for DNA |
-gapopen | Gap opening penalty | Number from 1.000 to 100.000 | 50.0 |
-gapextend | Gap extension penalty | Number from 0.100 to 10.000 | 5 |
Advanced qualifiers | Allowed values | Default | |
(none) |
The output is a standard EMBOSS alignment file.
The results can be output in one of several styles by using the command-line qualifier -aformat xxx, where 'xxx' is replaced by the name of the required format. Some of the alignment formats can cope with an unlimited number of sequences, while others are only for pairs of sequences.
The available multiple alignment format names are: unknown, multiple, simple, fasta, msf, trace, srs
The available pairwise alignment format names are: pair, markx0, markx1, markx2, markx3, markx10, srspair, score
See: http://www.uk.embnet.org/Software/EMBOSS/Themes/AlignFormats.html for further information on alignment formats.
The output report file contains descriptions of the positions where there is a mismatch in the alignment and shows the alignment. Where there is a mismatch in the alignment, the chosen base is written in uppercase.
######################################## # Program: merger # Rundate: Mon May 20 16:17:43 2002 # Report_file: stdout ######################################## #======================================= # # Aligned_sequences: 2 # 1: ECLACY # 2: ECLACA # Matrix: EDNAFULL # Gap_penalty: 50.0 # Extend_penalty: 5.0 # # Length: 3173 # Identity: 159/3173 ( 5.0%) # Similarity: 159/3173 ( 5.0%) # Gaps: 3014/3173 (95.0%) # Score: 795.0 # # #======================================= ECLACY 1 ttccagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaa 50 ECLACA 1 0 .................... until ...................... ECLACY 1301 cgcttagcggccccggcccgctttccctgctgcgtcgtcaggtgaatgaa 1350 ||||||||| ECLACA 1 gtgaatgaa 9 ECLACY 1351 gtcgcttaagcaatcaatgtcggatgcggcgcgacgcttatccgaccaac 1400 |||||||||||||||||||||||||||||||||||||||||||||||||| ECLACA 10 gtcgcttaagcaatcaatgtcggatgcggcgcgacgcttatccgaccaac 59 ECLACY 1401 atatcataacggagtgatcgcattgaacatgccaatgaccgaaagaataa 1450 |||||||||||||||||||||||||||||||||||||||||||||||||| ECLACA 60 atatcataacggagtgatcgcattgaacatgccaatgaccgaaagaataa 109 ECLACY 1451 gagcaggcaagctatttaccgatatgtgcgaaggcttaccggaaaaaaga 1500 |||||||||||||||||||||||||||||||||||||||||||||||||| ECLACA 110 gagcaggcaagctatttaccgatatgtgcgaaggcttaccggaaaaaaga 159 .................... until ...................... #--------------------------------------- # # ECLACY position base ECLACA position base Using # # #---------------------------------------
Program name | Description |
---|---|
cons | Creates a consensus from multiple alignments |
megamerger | Merge two large overlapping nucleic acid sequences |