Introduction to MIRA3

Bastien Chevreux

MIRA Version 3.4.1.1

Document revision $Id$

Table of Contents

1. What is MIRA?
2. What to read in this manual and where to start reading?
3. The MIRA quick tour
4. For which data sets to use MIRA and for which not
4.1. Genome de-novo
4.2. Genome mapping
4.3. ESTs / RNASeq
5. Any special features I might be interested in?
5.1. MIRA learns to discern non-perfect repeats, leading to better assemblies
5.2. MIRA has integrated editors for data from Sanger, 454, IonTorrent sequencing
5.3. MIRA lets you see why contigs end where they end
5.4. MIRA tags problematic decisions in hybrid assemblies
5.5. MIRA allows older finshing programs to cope with amount data in Solexa mapping projects
5.6. MIRA tags SNPs and other features, outputs result files for biologists
5.7. MIRA has ... much more
6. Versions, Licenses, Disclaimer and Copyright
6.1. Versions
6.2. License
6.2.1. MIRA
6.2.2. Documentation
6.3. Copyright
6.4. External libraries
7. Getting help / Mailing lists / Reporting bugs
8. Author
9. Miscellaneous
9.1. Citing MIRA
9.2. Postcards, gold and jewellery
 

Half of being smart is to know what you're dumb at.

 
 --Solomon Short

1.  What is MIRA?

MIRA is a multi-pass DNA sequence data assembler/mapper for whole genome and EST projects. MIRA assembles reads gained by

  • electrophoresis sequencing (aka Sanger sequencing)

  • 454 pyrosequencing (GS20, FLX or Titanium)

  • Ion Torrent

  • Solexa (Illumina) sequencing

  • (in development) Pacific Biosciences sequencing

into contiguous sequences (called contigs). One can use the sequences of different sequencing technologies either in a single assembly run (a true hybrid assembly) or by mapping one type of data to an assembly of other sequencing type (a semi-hybrid assembly (or mapping)) or by mapping a data against consensus sequences of other assemblies (a simple mapping).

The MIRA acronym stands for Mimicking Intelligent Read Assembly and the program pretty well does what its acronym says (well, most of the time anyway). It is the Swiss army knife of sequence assembly that I've used and developed during the past 14 years to get assembly jobs I work on done efficiently - and especially accurately. That is, without me actually putting too much manual work into it.

Over time, other labs and sequencing providers have found MIRA useful for assembly of extremely 'unfriendly' projects containing lots of repetitive sequences. As always, your mileage may vary.

2.  What to read in this manual and where to start reading?

At the last count, this manual had almost 200 pages and this might seem a little bit daunting. However, you very probably do not need to read everything.

You should read most of this introductionary chapter though: e.g.,

  • the part with the MIRA quick tour

  • the part which gives a quick overview for which data sets to use MIRA and for which not

  • the part which showcases different features of MIRA (lots of screenshots!)

  • where and how to get help if things don't work out as you expected

After that, reading should depend on the type of data you intend to work with: there are specific chapters for Sanger, 454, Solexa, IonTorrent and PacBio data, all of which containing an overview on how to prepare your data and how to launch MIRA for these data sets. There are also complete walkthroughs which exemplarily show from start to end one way of doing an assembly for a specific data set and what to do with the results of the assembly.

As the former named chapters are geared toward genome assemblies, there is also a chapter going into details on how to use MIRA for EST / RNASeq assemblies. Read that if you're into this kind of data.

As the previously cited chapters are more introductory in their nature, they do not go into the details of MIRA parametrisation. While MIRA has a comprehensive set of standard settings which should be suited for a majority of assembly tasks, the are more than 150 switches / parameters with which one can fine tune almost every aspect of an assembly. A complete description for each and every parameter and how to correctly set parameters for different use cases and sequencing technologies can be found in the reference chapter.

The chapter on working with results of MIRA should again be of general interest to everyone. It describes the structure of output directories and files and gives first pointers on what to find where. Also, converting results into different formats -- with and without filtering for specific needs -- is covered there.

As not every assembly project is simple, there is also a chapter with tipps on how to deal with projects which turn out to be "hard." It certainly helps if you at least skim through it even if you do not expect to have problems with your data ... it contains a couple of tricks on what one can see in result files as well as in temporary and log files which are not explained elsewhere.

As from time to time some general questions on sequencing are popping up on the MIRA talk mailing list, I have added a chapter with some general musings on what to consider when going into sequencing projects. This should be in no way a replacement for an exhaustive talk with a sequencing provider, but it can give a couple of hints on what to take care of.

There is also a FAQ chapter with some of the more frequently asked questions which popped up in the past few years.

Finally, there are also chapters covering some more technical aspects of MIRA: the MAF format and structure / content of the tmp directory have own chapters.

3.  The MIRA quick tour

Input can be in various formats like Staden experiment (EXP), Sanger CAF, FASTA, FASTQ or PHD file. Ancillary data containing additional information helpful to the assembly as is contained in, e.g. NCBI traceinfo XML files or Staden EXP files, is also honoured. If present, base qualities in phred style and SCF signal electrophoresis trace files are used to adjudicate between or even correct contradictory stretches of bases in reads by either the integrated automatic EdIt editor (written by Thomas Pfisterer) or the assembler itself.

MIRA was conceived especially with the problem of repeats in genomic data and SNPs in transcript (EST / RNASeq) data in mind. Considerable effort was made to develop a number of strategies -- ranging from standard clone-pair size restrictions to discovery and marking of base positions discriminating the different repeats / SNPs -- to ensure that repetitive elements are correctly resolved and that misassemblies do not occur.

The resulting assembly can be written in different standard formats like CAF, Staden GAP4 directed assembly, ACE, HTML, FASTA, simple text or transposed contig summary (TCS) files. These can easily be imported into numerous finishing tools or further evaluated with simple scripts.

The aim of MIRA is to build the best possible assembly by

  1. having a more or less full overview on the whole project at any time of the assembly, i.e. knowledge of almost all possible read-pairs in a project,

  2. using high confidence regions (HCRs) of several aligned read-pairs to start contig building at a good anchor point of a contig, extending clipped regions of reads on a 'can be justified' basis.

  3. using all available data present at the time of assembly, i.e., instead of relying on sequence and base confidence values only, the assembler will profit from trace files containing electrophoresis signals, tags marking possible special attributes of DNA, information on specific insert sizes of read-pairs etc.

  4. having 'intelligent' contig objects accept or refuse reads based on the rate of unexplainable errors introduced into the consensus

  5. learning from mistakes by discovering and analysing possible repeats differentiated only by single nucleotide polymorphisms. The important bases for discriminating different repetitive elements are tagged and used as new information.

  6. using the possibility given by the integrated automatic editor to correct errors present in contigs (and subsequently) reads by generating and verifying complex error hypotheses through analysis of trace signals in several reads covering the same area of a consensus,

  7. iteratively extending reads (and subsequently) contigs based on

    1. additional information gained by overlapping read pairs in contigs and

    2. corrections made by the automated editor.

MIRA was part of a bigger project that started at the DKFZ (Deutsches Krebsforschungszentrum, German Cancer Research Centre) Heidelberg in 1997: the "Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie" supported the PhD thesis of Thomas and myself by grant number 01 KW 9611. Beside an assembler to tackle difficult repeats, the grant also supported the automated editor / finisher EdIt package -- written by Thomas Pfisterer. The strength of MIRA and EdIt is the automatic interaction of both packages which produces assemblies with less work for human finishers to be done.

I'd like to thank everybody who reported bugs to me, pointed out problems, sent ideas and suggestions they encountered while using the predecessors. Please continue to do so, the feedback made this third version possible.

4.  For which data sets to use MIRA and for which not

As a general rule of thumb: if you have an organism with more than 100 to 150 megabases or more than 20 to 40 million reads, you might want to try other assemblers first.

4.1.  Genome de-novo

For genome assembly, the version 3 series of MIRA have been reported to work on projects with something like a million Sanger reads (~80 to 100 megabases at 10x coverage), five to ten million 454 Titanium reads (~100 megabases at 20x coverage) and 20 to 40 million Solexa reads (enough for de-novo of a bacterium or a small eukaryote with 76mers or 100mers).

Provided you have the memory, MIRA is expected to work in de-novo mode with

  • Sanger reads: 5 to 10 million

  • 454 reads: 5 to 15 million

  • Ion Torrent reads: 5 to 15 million

  • Solexa reads: 15 to 20 million

and "normal" coverages, whereas "normal" would be at no more than 50x to 70x for genome projects. Higher coverages will also work, but may create somewhat larger temporary files without heavy parametrisation. Lower coverages (<4x for Sanger, <10x for 454, < 10x for IonTorrent) also need special attention in the parameter settings.

4.2.  Genome mapping

As the complexity of mapping is a lot lower than de-novo, one can basically double (perhaps even triple) the number of reads compared to 'de-novo'. The limiting factor will be the amount of RAM though, and MIRA will also need lots of it if you go into eukaryotes.

The main limiting factor regarding time will be the number of reference sequences (backbones) you are using. MIRA being pedantic during the mapping process, it might be a rather long wait if you have more than 500 to 1000 reference sequences.

4.3.  ESTs / RNASeq

The default values for MIRA should allow it to work with many EST and RNASeq data sets, sometimes even from non-normalised libraries. For extreme coverage cases however (like, something with a lot of cases at and above 10k coverage), one would perhaps want to resort to data reduction routines before feeding the sequences to MIRA.

On the other hand, recent developments of MIRA were targeted at making de-novo RNASeq assembly of non-normalised libraries liveable, and indeed I now regularly use MIRA for data sets with up to 50 million Illumina 100bp reads.

5.  Any special features I might be interested in?

A few perhaps.

[Note]Note

The screenshots in this section show data from assemblies produced with MIRA, but the visualisation itself is done in a finishing program named gap4.

Some of the screenshots were edited for showing a special feature of MIRA. E.g., in the screenshots with Solexa data, quite some reads were left out of the view pane as else -- due to the amount of data -- these screenshots would need several pages for a complete printout.

5.1.  MIRA learns to discern non-perfect repeats, leading to better assemblies

MIRA is an iterative assembler (it works in several passes) and acts a bit like a child when exploring the world: it explores the assembly space and is specifically parametrised to allow a couple of assembly errors during the first passes. But after each pass some routines (the "parents", if you like) check the result, searching for assembly errors and deduce knowledge about specific assemblies MIRA should not have ventured into. MIRA will then prevent these errors to re-occur in subsequent passes.

As an example, consider the following multiple alignment:

Figure 1. How MIRA learns from misassemblies (1). Multiple alignment after 1st pass with an obvious assembly error, notice the clustered columns discrepancies. Two slightly different repeats were assembled together.

How MIRA learns from misassemblies (1). Multiple alignment after 1st pass with an obvious assembly error, notice the clustered columns discrepancies. Two slightly different repeats were assembled together.

These kind of errors can be easily spotted by a human, but are hard to prevent by normal alignment algorithms as sometimes there's only one single base column difference between repeats (and not several as in this example).

MIRA spots these things (even if it's only a single column), tags the base positions in the reads with additional information and then will use that information in subsequent passes. The net effect is shown in the next two figures:

Figure 2.  Multiple alignment after last pass where assembly errors from previous passes have been resolved (1st repeat site)

Multiple alignment after last pass where assembly errors from previous passes have been resolved (1st repeat site)

Figure 3.  Multiple alignment after last pass where assembly errors from previous passes have been resolved (2nd repeat site)

Multiple alignment after last pass where assembly errors from previous passes have been resolved (2nd repeat site)

The ability of MIRA to learn and discern non-identical repeats from each other through column discrepancies is nothing new. Here's the link to a paper from a talk I had at the German Conference on Bioinformatics in 1999: http://www.bioinfo.de/isb/gcb99/talks/chevreux/

I'm sure you'll recognise the basic principle in figures 8 and 9. The slides from the corresponding talk also look very similar to the screenshots above:

Figure 4.  Slides presenting the repeat locator at the GCB 99

Slides presenting the repeat locator at the GCB 99

You can get the talk with these slides here: http://chevreux.org/dkfzold/gcb99/bachvortrag_gcb99.ppt

5.2.  MIRA has integrated editors for data from Sanger, 454, IonTorrent sequencing

Since the first versions in 1999, the EdIt automatic Sanger sequence editor from Thomas Pfisterer has been integrated into MIRA.

Figure 5.  Slides presenting the Edit automatic Sanger editor at the GCB 99

Slides presenting the Edit automatic Sanger editor at the GCB 99

The routines use a combination of hypothesis generation/testing together with neural networks (trained on ABI and ALF traces) for signal recognition to discern between base calling errors and true multiple alignment differences. They go back to the trace data to resolve potential conflicts and eventually recall bases using the additional information gained in a multiple alignment of reads.

Figure 6.  Sanger assembly without EdIt automatic editing routines. The bases with blue background are base calling errors.

Sanger assembly without EdIt automatic editing routines. The bases with blue background are base calling errors.

Figure 7.  Sanger assembly with EdIt automatic editing routines. Bases with pink background are corrections made by EdIt after assessing the underlying trace files (SCF files in this case). Bases with blue background are base calling errors where the evidence in the trace files did not show enough evidence to allow an editing correction.

Sanger assembly with EdIt automatic editing routines. Bases with pink background are corrections made by EdIt after assessing the underlying trace files (SCF files in this case). Bases with blue background are base calling errors where the evidence in the trace files did not show enough evidence to allow an editing correction.

With the introduction of 454 reads, MIRA also got in 2007 specialised editors to search and correct for typical 454 sequencing problems like the homopolymer run over-/undercalls. These editors are now integrated into MIRA itself and are not part of EdIt anymore.

While not being paramount to the assembly quality, both editors provide additional layers of safety for the MIRA learning algorithm to discern non-perfect repeats even on a single base discrepancy. Furthermore, the multiple alignments generated by these two editors are way more pleasant to look at (or automatically analyse) than the ones containing all kind of gaps, insertions, deletions etc.pp.

Figure 8.  454 assembly without 454 automatic editing routines

454 assembly without 454 automatic editing routines

Figure 9.  454 assembly with 454 automatic editing routines

454 assembly with 454 automatic editing routines

With introduction of PacBio strobed reads, MIRA also got an editor to handle "elastic dark inserts" (stretches of unread bases where the length is known only approximately). How this editor works is explained in the chapter on PacBio data, but in essence it allows to transform this:

Figure 10.  Multiple alignmen of PacBio elastic dark inserts without automatic editing

Multiple alignmen of PacBio elastic dark inserts without automatic editing

into this:

Figure 11.  Multiple alignment of PacBio elastic dark inserts with automatic editing

Multiple alignment of PacBio elastic dark inserts with automatic editing

5.3.  MIRA lets you see why contigs end where they end

A very useful feature for finishing are hash frequency (HAF) tags which MIRA sets in the assembly. Provided your finishing editor understands those tags (gap4, gap5 and consed are fine but there may be others), they'll give you precious insight where you might want to be cautious when joining to contigs or where you would need to perform some primer walking. MIRA colourises the assembly with the HAF tags to show repetitiveness.

You will need to read about the HAF tags in the reference manual, but in a nutshell: the HAF5, HAF6 and HAF7 tags tell you potentially have repetitive to very repetitive read areas in the genome, while HAF2 tags will tell you that these areas in the genome have not been covered as well as they should have been.

As an example, the following figure shows the coverage of a contig.

Figure 12.  Coverage of a contig.

Coverage of a contig.

The question is now: why did MIRA stop building this contig on the left end (left oval) and why on the right end (right oval).

Looking at the HAF tags in the contig, the answer becomes quickly clear: the left contig end has HAF5 tags in the reads (shown in bright red in the following figure). This tells you that MIRA stopped because it probably could not unambiguously continue building this contig. Indeed, if you BLAST the sequence at the NCBI, you will find out that this is an rRNA area of a bacterium, of which bacteria normally have several copies in the genome:

Figure 13.  HAF5 tags (reads shown with red background) covering a contig end show repetitiveness as reason for stopping a contig build.

HAF5 tags (reads shown with red background) covering a contig end show repetitiveness as reason for stopping a contig build.

The right end of the same contig however ends in HAF3 tags (normal coverage, bright green in the next figure) and even HAF2 tags (below average coverage, pale green in the next image). This tells you MIRA stopped building the contig at this place simply because there were no more reads to continue. This is a perfect target for primer walking if you want to finish a genome.

Figure 14.  HAF2 tags covering a contig end show that no more reads were available for assembly at this position.

HAF2 tags covering a contig end show that no more reads were available for assembly at this position.

5.4.  MIRA tags problematic decisions in hybrid assemblies

Many people combine Sanger & 454 -- or nowadays more 454 & Solexa -- to improve the sequencing quality of their project through two (or more) sequencing technologies. To reduce time spent in finishing, MIRA automatically tags those bases in a consensus of a hybrid assembly where reads from different sequencing technologies severely contradict each other.

The following example shows a hybrid 454 / Solexa assembly where reads from 454 (highlighted read names in following figure) were not sure whether to have one or two "G" at a certain position. The consensus algorithm would have chosen "two Gs" for 454, obviously a wrong decision as all Solexa reads at the same spot (the reads which are not highlighted) show only one "G" for the given position. While MIRA chose to believe Solexa in this case, it tagged the position anyway in case someone choses to check these kind of things.

Figure 15.  A "STMS" tag (Sequencing Technology Mismatch Solved, the black square base in the consensus) showing a potentially difficult decision in a hybrid 454 / Solexa de-novo assembly.

A "STMS" tag (Sequencing Technology Mismatch Solved, the black square base in the consensus) showing a potentially difficult decision in a hybrid 454 / Solexa de-novo assembly.

This works also for other sequencing technology combinations or in mapping assemblies. The following is an example in a hybrid Sanger / 454 project where by pure misfortune, all Sanger reads have a base calling error at a given position while the 454 reads show the true sequence.

Figure 16.  A "STMU" tag (Sequencing Technology Mismatch Unresolved, light blue square in the consensus at lower end of large oval) showing a potentially difficult decision in a hybrid Sanger / 454 mapping assembly.

A "STMU" tag (Sequencing Technology Mismatch Unresolved, light blue square in the consensus at lower end of large oval) showing a potentially difficult decision in a hybrid Sanger / 454 mapping assembly.

5.5.  MIRA allows older finshing programs to cope with amount data in Solexa mapping projects

Quality control is paramount when you do mutation analysis for biologists: I know they'll be on my doorstep the very next minute they found out one of the SNPs in the resequencing data wasn't a SNP, but a sequencing artefact. And I can understand them: why should they invest -- per SNP -- hours in the wet lab if I can invest a couple of minutes to get them data false negative rates (and false discovery rates) way below 1%? So, finishing and quality control for any mapping project is a must.

Both gap4 and consed start to have a couple of problems when projects have millions of reads: you need lots of RAM and scrolling around the assembly gets a test to your patience. Still, these two assembly finishing programs are amongst the better ones out there, although gap5 starts to quickly arrive in a state in which it allows itself to substitute to gap4.

So, MIRA reduces the number of reads in Solexa mapping projects without sacrificing information on coverage. The princible is pretty simple: for 100% matching reads, MIRA tracks coverage of every reference base and creates long synthetic, coverage equivalent reads (CERs) in exchange for the Solexa reads. Reads that do not match 100% are kept as own entities, so that no information gets lost. The following figure illustrates this:

Figure 17.  Coverage equivalent reads (CERs) explained.

Left side of the figure: a conventional mapping with eleven reads of size 4 against a consensus (in uppercase). The inversed base in the lowest read depicts a sequencing error.

Right side of the figure: the same situation, but with coverage equivalent reads (CERs). Note that there are less reads, but no information is lost: the coverage of each reference base is equivalent to the left side of the figure and reads with differences to the reference are stil present.

Coverage equivalent reads (CERs) explained. Left side of the figure: a conventional mapping with eleven reads of size 4 against a consensus (in uppercase). The inversed base in the lowest read depicts a sequencing error. Right side of the figure: the same situation, but with coverage equivalent reads (CERs). Note that there are less reads, but no information is lost: the coverage of each reference base is equivalent to the left side of the figure and reads with differences to the reference are stil present.

This strategy is very effective in reducing the size of a project. As an example, in a mapping project with 9 million Solexa 36mers, MIRA created a project with 1.7m reads: 700k CER reads representing ~8 million 100% matching Solexa reads, and it kept ~950k mapped reads as they had ≥ mismatch (be it sequencing error or true SNP) to the reference. A reduction of 80%, and numbers for mapping projects with Solexa 100bp reads are in a similar range.

Also, mutations of the resequenced strain now really stand out in the assembly viewer as the following figure shows:

Figure 18.  Coverage equivalent reads let SNPs become very visible in assembly viewers

Coverage equivalent reads let SNPs become very visible in assembly viewers

5.6.  MIRA tags SNPs and other features, outputs result files for biologists

Want to assemble two or several very closely related genomes without reference, but finding SNPs or differences between them?

Tired of looking at some text output from mapping programs and guessing whether a SNP is really a SNP or just some random junk?

MIRA tags all SNPs (and other features like missing coverage etc.) it finds so that -- when using a finishing viewer like gap4 or consed -- one can quickly jump from tag to tag and perform quality control. This works both in de-novo assembly and in mapping assembly, all MIRA needs is the information which read comes from which strain.

The following figure shows a mapping assembly of Solexa 36mers against a bacterial reference sequence, where a mutant has an indel position in an gene:

Figure 19.  "SROc" tag (Snp inteR Organism on Consensus) showing a SNP position in a Solexa mapping assembly.

"SROc" tag (Snp inteR Organism on Consensus) showing a SNP position in a Solexa mapping assembly.

Other interesting places like deletions of whole genome parts are also directly tagged by MIRA and noted in diverse result files (and searchable in assembly viewers):

Figure 20.  "MCVc" tag (Missing CoVerage in Consenus, dark red stretch in figure) showing a genome deletion in Solexa mapping assembly.

"MCVc" tag (Missing CoVerage in Consenus, dark red stretch in figure) showing a genome deletion in Solexa mapping assembly.

[Note]Note
For bacteria -- and if you use annotated GenBank files as reference sequence -- MIRA will also output some nice lists directly usable (in Excel) by biologists, telling them which gene was affected by what kind of SNP, whether it changes the protein, the original and the mutated protein sequence etc.pp.

5.7.  MIRA has ... much more

  • Extensive possibilities to clip data if needed: by quality, by masked bases, by A/T stretches, by evidence from other reads, ...

  • Routines to re-extend reads into clipped parts if multiple alignment allows for it.

  • Read in ancillary data in different formats: EXP, NCBI TRACEINFO XML, SSAHA2, SMALT result files and text files.

  • Detection of chimeric reads.

  • Pipeline to discover SNPs in ESTs from different strains (miraSearchESTSNPs)

  • Support for many different of input and output formats (FASTA, EXP, FASTQ, CAF, MAF, ...)

  • Automatic memory management (when RAM is tight)

  • Over 150 parameters to tune the assembly for a lot of use cases, many of these parameters being tunable individually depending on sequencing technology they apply to.

6.  Versions, Licenses, Disclaimer and Copyright

6.1.  Versions

There are two kind of versions for MIRA that can be compiled form source files: production and development.

Production versions are from the stable branch of the source code. These versions are available for download from SourceForge.

Development versions are from the development branch of the source tree. These are also made available to the public and should be compiled by users who want to test out new functionality or to track down bugs or errors that might arise at a given location. Release candidates (rc) also fall into the development versions: they are usually the last versions of a given development branch before being folded back into the production branch.

6.2.  License

6.2.1.  MIRA

MIRA has been put under the GPL version 2.

This program is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2 of the License, or (at your option) any later version.

This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.

You should have received a copy of the GNU General Public License along with this program; if not, write to the Free Software Foundation, Inc., 51 Franklin Street, Fifth Floor, Boston, MA 02110-1301, USA

You may also visit http://www.opensource.org/licenses/gpl-2.0.php at the Open Source Initiative for a copy of this licence.

6.2.2.  Documentation

The documentation pertaining to MIRA is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ or send a letter to Creative Commons, 171 Second Street, Suite 300, San Francisco, California, 94105, USA.

6.3.  Copyright

© 1997-2000 Deutsches Krebsforschungszentrum Heidelberg -- Dept. of Molecular Biophysics and Bastien Chevreux (for MIRA) and Thomas Pfisterer (for EdIt)

© 2001-2011 Bastien Chevreux.

All rights reserved.

6.4.  External libraries

MIRA uses the excellent Expat library to parse XML files. Expat is Copyright © 1998, 1999, 2000 Thai Open Source Software Center Ltd and Clark Cooper as well as Copyright © 2001, 2002 Expat maintainers.

See http://www.libexpat.org/ and http://sourceforge.net/projects/expat/ for more information on Expat.

7.  Getting help / Mailing lists / Reporting bugs

Please try to find an answer to your question by first reading the documents provided with the MIRA package (FAQs, READMEs, usage guide, guides for specific sequencing technologies etc.). It's a lot, but then again, they hopefully should cover 90% of all questions.

If you have a tough nut to crack or simply could not find what you were searching for, you can subscribe to the MIRA talk mailing list and send in your question (or comment, or suggestion), see http://www.chevreux.org/mira_mailinglists.html for more information on that. Now that the number of subscribers has reached a good level, there's a fair chance that someone could answer your question before I have the opportunity or while I'm away from mail for a certain time.

[Note]Note

Please very seriously consider using the mailing list before mailing me directly. Every question which can be answered by participants of the list is time I can invest in development and documentation of MIRA. I have a day job as bioinformatician which has nothing to do with MIRA and after work hours are rare enough nowadays.

Furthermore, Google indexes the mailing list and every discussion / question asked on the mailing list helps future users as they show up in Google searches.

Only mail me directly (bach@chevreux.org) if you feel that there's some information you absolutely do not want to share publicly.

[Note]Note
Subscribing to the list before sending mails to it is necessary as messages from non-subscribers will be stopped by the system to keep the spam level low.

To report bugs or ask for new features, please use the new ticketing system at: http://sourceforge.net/apps/trac/mira-assembler/. This ensures that requests do not get lost and you get the additional benefit to automatically know when a bug has been fixed (there won't be separate emails sent, that's what bug trackers are there for).

Finally, new or intermediate versions of MIRA will be announced on the separate MIRA announce mailing list. Traffic is very low there as the only one who can post there is me. Subscribe if you want to be informed automatically on new releases of MIRA.

8.  Author

Bastien Chevreux (mira):

WWW: http://www.chevreux.org/

MIRA can use automatic editing routines for Sanger sequences which were written by Thomas Pfisterer (EdIt):

9.  Miscellaneous

9.1.  Citing MIRA

Please use these citations:

For mira

Chevreux, B., Wetter, T. and Suhai, S. (1999): Genome Sequence Assembly Using Trace Signals and Additional Sequence Information. Computer Science and Biology: Proceedings of the German Conference on Bioinformatics (GCB) 99, pp. 45-56.

For miraSearchESTSNPs (was named miraEST in earlier times)

Chevreux, B., Pfisterer, T., Drescher, B., Driesel, A. J., Müller, W. E., Wetter, T. and Suhai, S. (2004): Using the miraEST Assembler for Reliable and Automated mRNA Transcript Assembly and SNP Detection in Sequenced ESTs. Genome Research, 14(6)

9.2.  Postcards, gold and jewellery

If you find this software useful, please send the author a postcard. If postcards are not available, a treasure chest full of Spanish doubloons, gold and jewellery will do nicely, thank you.