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Explicit definition of file types. It is possible to explicitly tell MIRA the type of a file even if said file does not have a 'standard' naming scheme. This does of course work also with directories or wildcard characters.

In the following example, the first line will load all files from mydirectory as FASTQ while the second line loads just. It is entirely possible although not really sensible , to give contradicting information to MIRA by using a different explicit file type than one would guess from the standard postfix.

In this case, the explicit type takes precedence over the automatic type. Note that the above does not make any kind of file conversion, file. The text technology is not a technology per se, but should be used for sequences which are not coming from sequencing machines like, e.

This is basically the lazy way to tell MIRA that the data in the corresponding readgroup consists of paired reads and that you trust it will find out the correct values. Defines the minimum and maximum size of "good" DNA templates in the library prep for this read group. This defines at which distance the two reads of a pair are to be expected in a contig, a very useful information for an assembler to resolve repeats in a genome or different splice variants in transcriptome data.

If the term infoonly is present, then MIRA will pass the information on template sizes in result files, but will not use it for any decision making during de-novo or mapping assembly.

If the term autorefine is present, MIRA will start the assembly with the given size information but switch to refined value computed from observed distances in an assembly.

However, please note that the size values can never be expanded, only shrunk. It is therefore advisable to use generous bounds when using the autorefine feature.

For de-novo assemblies however you should not use infoonly except in very rare cases where you know what you do. When using a paired-end or mate-pair sequencing strategy, two sequences are generated for the ends of each DNA template see sidebar above: That is, if one has a library with 6kb fragments, one knows that the outer ends of the two reads will be approximately 6kb apart, like so:.

Sequencing labs will try their best to get these two sequences from DNA templates which comply to a given length specification. But as this is chemistry and wet lab, things must be seen with a certain uncertainty and therefore the DNA templates generated are not exactly of the specified size e. Allowed placement codes are:. This is typically used for unpaired libraries sometimes called shotgun libraries , but may be also useful for, e. This is the usual placement code for Sanger paired-end protocols as well as Illumina paired-end.

Less frequently used in IonTorrent paired-end sequencing. Segments of a template are all placed in the same direction, the segment order in the contig follows segment ordering of the reads. Segments of a template are all placed in the same direction, the segment order in the contig is reversed compared to segment ordering of the reads. If the term infoonly is present, then MIRA will pass the information on segment placement in result files, but will not use it for any decision making during de-novo assembly or mapping assembly.

Defines the naming scheme reads are following to indicate the DNA template they belong to. Allowed naming schemes are: If not defined, the defaults are sanger for Sanger sequencing data, while solexa for Solexa, and Ion Torrent. Read naming is a long story with lots of historical gotchas: Unsurprisingly, several "standards" emerged over time. See also XKCD entry on proliferating standards. In a nutshell and probably over-simplified:. This is to allow multiple sequencing of a fragment, particularly common with Sanger capillary data e.

Allows to rename reads on the fly while loading data by searching each read name for a given prefix string and, if found, replace it with a given replacement string. This is most useful for systems like Illumina or PacBio which generate quite long read names which, in the end, are either utterly useless for an end user or are even breaking older programs which have a length restriction on read names. This ranges from fine-tuning assemblies to setting parameters in a way so that MIRA is suited also for very special assembly cases.

Some parameters one can set in MIRA somehow belong together. MIRA uses parameter groups to keep parameters together which somehow belong together. The parameters of the different parameter groups are described in detail a bit later in this manual.

With the introduction of new sequencing technologies, MIRA also had to be able to set values that allow technology specific behaviour of algorithms. One simple example for this could be the minimum length a read must have to be used in the assembly. For Sanger sequences, having this value to be meaning a read should have at least unclipped bases would be a very valid, albeit conservative choice.

To allow very fine grained behaviour, especially in hybrid assemblies, and to prevent the explosion of parameter names, MIRA knows two categories of parameters:. Example for this would be the minimum length of a read like for Sanger reads and for FLX reads. As example, a manifest using technology dependent and independent parameters could look like this:. For MIRA, this means a number of parameters should apply to the assembly as whole, while others apply to the sequencing data itself MIRA dumps the parameters it is running with at the beginning of an assembly and it makes it clear there which parameters are "global" and which parameters apply to single technologies.

Here is as example a part of the output of used parameters that MIRA will show when started with and Illumina Solexa data:. You can see the two different kind of settings that MIRA uses: How would one set a minimum read length of 40 and not enforce presence of base qualities for Sanger reads, but for reads a minimum read length of 30 and enforce base qualities? Alternatively you can use a backslash at the end of a parameter line to indicate that the next line is a continuing line, like so:.

For example, it would not make sense to try and set different number of assembly passes for each technology like in. Beside being contradictory, this makes not really sense. MIRA will complain about cases like these.

Please note that it is also perfectly legal to decompose the switches so that they can be used more easily in scripted environments notice the multiple -AL in some sections of the following example:. For some parameters, the order of appearance in the parameter lines of the manifest is important.

This is because the quick parameters are realised internally as a collection of extended parameters that will overwrite any previously manually set extended parameters. It is generally a good idea to place quick parameters in the order as described in this documentation, that is: These are modifier switches for genome data that is deemed to be highly repetitive.

Usage recommendations for 'simple' lower eukaryotes: Usage recommendations for lower eukaryotes with complex repeats: Repeats occurring more often will not be resolved, but using the debris information one can recover affected reads and use these with harsh data reduction algorithms e.

Switches off clipping options for given sequencing technologies. Technologies can be sanger , , iontor , solexa or solid. Multiple entries separated by comma. Switch off and Solexa but keep eventually keep Sanger clipping: General options control the type of assembly to be performed and other switches not belonging anywhere else. Master switch to set the number of threads used in different parts of MIRA.

A value of 0 tells MIRA to set this to the number of available physical cores on the machine it runs on. That is, hyperthreaded "cores" are not counted in as using these would cause a tremendous slowdown in the heavy duty computation parts.

In case MIRA cannot find out the number of cores, the fall-back value is 2. Besides, at the latest when the Smith-Watermans run in parallel, this could not be easily avoided at all.

If this is not available, the functionality is switched off. The automatic memory management can only work if there actually is unused system memory. It's not a wonder switch which reduces memory consumption. In tight memory situations, memory management has no effect and the algorithms fall back to minimum table sizes. This means that the effective size in memory can grow larger than given in the memory management parameters, but then MIRA will try to keep the additional memory requirements to a minimum.

A value of 0 means that MIRA does not try keep a fixed upper limit. If automatic memory management is used see above , this number works a bit like [-GE: EST assembly is a three step process, each with different settings to the assembly engine, with the result of each step being saved to disk.

If results of previous steps are present in a directory, one can easily "play around" with different setting for subsequent steps by reusing the results of the previous steps and directly starting with step two or three. Controls whether date and time are printed out during the assembly.

Suppressing it is not useful in normal operation, only when debugging or benchmarking. Default is dependent of the sequencing technology and assembly quality level. Defines how many iterations of the whole assembly process are done.

As a special use case, a value of 0 will let MIRA just run the following tasks: The resulting reads can then be found as MAF file in the checkpoint directory; the read repeat information in the info directory.

Defines whether the skim algorithm and with it also the recalculation of Smith-Waterman alignments is called in-between each main pass. If set to no , skimming is done only when needed by the workflow: Setting this option to yes is highly recommended, setting it to no only for quick and dirty assemblies.

Defines the maximum number of times a contig can be rebuilt during a main assembly passes [-AS: Defines how many contigs are maximally built in each pass. A value of 0 stands for 'unlimited'. Default is is currently yes. Tells MIRA to use coverage information accumulated over time to more accurately pinpoint reads that are in repetitive regions. This option says this: Default is dependent of the sequencing technology, currently for Sanger and for and Ion Torrent.

A coverage must be at least this number of bases higher than [-AS: Default is currently always no as these algorithms were supplanted by better ones in MIRA 4. When set to yes , MIRA will analyse coverage of contigs built at a certain stage of the assembly and estimate an average expected coverage of reads for contigs. This value will be used in subsequent passes of the assembly to ensure that no part of the contig gets significantly more read coverage of reads that were previously identified as repetitive than the estimated average coverage allows for.

It is expected to be useful for Sanger and sequences. Usage of this switch with Solexa and Ion Torrent data is currently not recommended. It is a real improvement to disentangle repeats, but has the side-effect of creating some "contig debris" small and low coverage contigs, things you normally can safely throw away as they are representing sequence that already has enough coverage. This switch must be set to no for EST assembly, assembly of transcripts etc. It is recommended to also switch this off for mapping assemblies.

Assemblies with 5 passes or more should set the value to the number of passes minus 2. Reads that bring the coverage above the threshold will be rejected from that specific place in the contig and either be built into another copy of the repeat somewhere else or end up as contig debris.

Please note that the lower [-AS: A spoiler can be either a chimeric read or it is a read with long parts of unclipped vector sequence still included that was too long for the [-CL: A spoiler typically prevents contigs to be joined, MIRA will cut them back so that they represent no more harm to the assembly.

Recommended for assemblies of mid- to high-coverage genomic assemblies, not recommended for assemblies of ESTs as one might loose splice variants with that. A minimum number of two assembly passes [-AS: Defines whether the spoiler detection algorithms are run only for the last pass or for all passes [-AS: Default is dependent of the sequencing technology.

Defines the minimum length that reads must have to be considered for the assembly. Shorter sequences will be filtered out at the beginning of the process and won't be present in the final project.

Default is dependent of the sequencing technology and the [--job] parameter. In EST assemblies, it's currently 2 for all sequencing technologies. Defines the minimum number of reads a contig must have before it is built or saved by MIRA. Overlap clusters with less reads than defined will not be assembled into contigs but reads in these clusters will be immediately transferred to debris.

This parameter is useful to considerably reduce assembly time in large projects with millions of reads like in Solexa projects where a lot of small "junk" contigs with contamination sequence or otherwise uninteresting data may be created otherwise.

When set to yes, MIRA will stop the assembly if any read has no quality values loaded. MIRA has two different pathfinder algorithms it chooses from to find its way through the more or less complete set of possible sequence overlaps: The genomic looks a bit into the future of the assembly and tries to stay on safe grounds using a maximum of information already present in the contig that is being built.

The EST version on the contrary will directly jump at the complex cases posed by very similar repetitive sequences and try to solve those first and is willing to fall back to first-come-first-served when really bad cases like, e.

Generally, the genomic pathfinder will also work quite well with EST sequences but might get slowed down a lot in pathological cases , while the EST algorithm does not work so well on genomes. If in doubt, leave on yes for genome projects and set to no for EST projects. Another important switch if you plan to assemble non-normalised EST libraries, where some ESTs may reach coverages of several hundreds or thousands of reads.

This switch lets MIRA save a lot of computational time when aligning those extremely high coverage areas but only there , at the expense of some accuracy.

Defines the number of potential partners a read must have for MIRA switching into emergency search stop mode for that read. Defines whether there is an upper limit of time to be used to build one contig.

Set this to yes in EST assemblies where you think that extremely high coverages occur. Less useful for assembly of genomic sequences.

Default is yes for mapping assemblies with Illumina data, no otherwise. When set to 'yes', MIRA will use a two stage mapping process which bootstraps an intermediate backbone reference sequence and greatly improves mapping accuracy at indel sites. Default is dependent on assembly quality level chosen: When assembling against backbones, this parameter defines the pass iteration see [-AS: In the passes preceding this number, the non-backbone reads will be assembled together as if no backbones existed.

This allows MIRA to correctly spot repetitive stretches that differ by single bases and tag them accordingly. Note that full assemblies are considerably slower than mapping assemblies, so be careful with this when assembling millions of reads. If backbones are a different strain, then set [-SB: Parameter for the internal sectioning size of the backbone to compute optimal alignments.

When set to 0, MIRA will compute optimal values from the data loaded. Should be set to length of the longest read. When set to 'yes', MIRA will trim back reads at end of contigs which outgrow the reference sequence so that boundaries of the reference and the mapped reads align perfectly. That is, the mapping does not perform a sequence extension. Standard mapping assembly mode of the assembler is to map available reads to a backbone and discard reads that do not fit.

If set to 'yes', MIRA will use reads that did not map to the backbone s to make new contigs if possible. This means, that in terms of memory consumption and speed, this switch combines the worst of both worlds. Default is dependent of the sequencing technology used: MIRA expects the sequences it is given to be quality clipped. During the assembly though, it will try to extend reads into the clipped region and gain additional coverage by analysing Smith-Waterman alignments between reads that were found to be valid.

Only the right clip is extended though, the left clip most of the time containing sequencing vector is never touched. Default is dependent of the sequencing technology used.

Only takes effect when [-DP: The read extension routines use a sliding window approach on Smith-Waterman alignments. This parameter defines the window length. This parameter defines the first pass in which the read extension routines are called.

The default of 0 tells MIRA to extend the reads the first time before the first assembly pass. This parameter defines the last pass in which the read extension routines are called.

The default of 0 tells MIRA to extend the reads the last time before the first assembly pass. Every option in this section can be set individually for every sequencing technology, giving a very fine grained control on how reads are clipped for each technology. While performing the clip of screened vector sequences, MIRA will look if it can merge larger chunks of sequencing vector bases that are a maximum of [-CL: While performing the clip of screened vector sequences at the start of a sequence, MIRA will allow up to this number of non-vector bases in front of a vector stretch.

While performing the clip of screened vector sequences at the end of a sequence, MIRA will allow up to this number of non-vector bases behind a vector stretch.

MIRA will try to identify possible sequencing vector relics present at the start of a sequence and clip them away.

These relics are usually a few bases long and were not correctly removed from the sequence in data preprocessing steps of external programs. You might want to turn off this option if you know or think that your data contains a lot of repeats and the option below to fine tune the clipping behaviour does not give the expected results. You certainly want to turn off this option in EST assemblies as this will quite certainly cut back and thus hide different splice variants.

But then make certain that you pre-processing of Sanger data sequencing vector removal is good, other sequencing technologies are not affected then. The clipping of possible vector relics option works quite well.

Unfortunately, especially the bounds of repeats or differences in EST splice variants sometimes show the same alignment behaviour than possible sequencing vector relics and could therefore also be clipped.

To refrain the vector clipping from mistakenly clip repetitive regions or EST splice variants, this option puts an upper bound to the number of bases a potential clip is allowed to have. If the number of bases is below or equal to this threshold, the bases are clipped. If the number of bases exceeds the threshold, the clip is NOT performed. Setting the value to 0 turns off the threshold, i.

This will let MIRA perform its own quality clipping before sequences are entered into the assembly. The clip function performed is a sequence end window quality clip with back iteration to get a maximum number of bases as useful sequence.

Note that the bases clipped away here can still be used afterwards if there is enough evidence supporting their correctness when the option [-DP: The windowing algorithm works pretty well for Sanger, but apparently does not like type data. It's advisable to not switch it on for Beside, the quality clipping algorithm performs a pretty decent albeit not perfect job, so for genomic data not! ESTs , it is currently recommended to use a combination of [-CL: This is the minimum quality bases in a window require to be accepted.

Please be cautious not to take too extreme values here, because then the clipping will be too lax or too harsh. Values below 15 and higher than are not recommended. This is the length of a window in bases for the quality clip. This option allows to clip reads that were not correctly preprocess and have unclipped bad quality stretches that might prevent a good assembly. MIRA will search the sequence in forward direction for a stretch of bases that have in average a quality less than a defined threshold and then set the right quality clip of this sequence to cover the given stretch.

Defines the minimum average quality a given window of bases must have. If this quality is not reached, the sequence will be clipped at this position. Defines the length of the window within which the average quality of the bases are computed. It is generally not a good idea to use mask bases to remove unwanted portions of a sequence, the EXP file format and the NCBI traceinfo format have excellent possibilities to circumvent this. MIRA will look at the start and end of each sequence to see whether there are masked bases that should be 'clipped'.

While performing the clip of masked bases, MIRA will look if it can merge larger chunks of masked bases that are a maximum of [-CL: While performing the clip of masked bases at the start of a sequence, MIRA will allow up to this number of unmasked bases in front of a masked stretch.

While performing the clip of masked bases at the end of a sequence, MIRA will allow up to this number of unmasked bases behind a masked stretch. This will let MIRA perform a 'clipping' of bases that are in lowercase at the front end of a sequence, leaving only the uppercase sequence.

Useful when handling data that does not have ancillary data in XML format. This will let MIRA perform a 'clipping' of bases that are in lowercase at the back end of a sequence, leaving only the uppercase sequence. Poly-A stretches in forward reads and poly-T stretches in reverse reads get either clipped or tagged here see [-CL: The assembler will not use these stretches for finding overlaps, but it will use these to discern and disassemble different 3' UTR endings.

Default is yes but takes effect only if [-CL: The tags provide additional information for MIRA to discern between different 3' UTR endings and alse a good visual anchor when looking at the assembly with different programs. Only takes effect when [-CP: Defines the number of 'A' in forward direction or 'T' in reverse direction must be present to be considered a poly-A sequence stretch. Only takes effect when [-CL: Defines the maximum number of errors allowed in the potential poly-A sequence stretch.

The distribution of these errors is not important. Defines the number of bases from the end of a sequence if masked: Defines whether MIRA should search and clip known sequencing technology specific sequencing adaptors. As the list of known adaptors changes quite frequently, the best place to get a list of known adaptors by MIRA is by looking at the text files in the program sources: If on, ensures a minimum left clip on each read according to the parameters in [-CL: If on, ensures a minimum right clip on each read according to the parameters in [-CL: When this parameter is set, MIRA will use that info to cut back chimeras to their longest non-chimeric length.

When this parameter is set, MIRA will use that info to cut back junk in reads. It is currently suggested to leave this parameter switched off as the routines seem to be a bit too "trigger happy" and also cut back perfectly valid sequences. Default is is dependent on --job quality: Switched off for EST assemblies but one might want to switch it on sometimes. This implements a pretty powerful strategy to ensure a good "high confidence region" HCR in reads, basically eliminating Note that one still must ensure that sequencing vectors Sanger or adaptor sequences , Solexa ion Torrent are "more or less" clipped prior to assembly.

Solexa data has a pretty awful problem with in some reads when a GGCxG motif occurs read more about it in the chapter on Solexa data. MIRA knows about this problem and can look for it in Solexa reads during the proposed end clipping and further clip back the reads, greatly minimising the impact of this problem. Default is is dependent on technology and quality in the --job switch: Ion Torrent has at the moment 17 , but this may change in the future to somewhat higher values.

This parameter defines the minimum number of bases at each end of a read that should be free of any sequencing errors. Note that the algorithm is based on SKIM hashing see below , and compares hashes of all reads with each other. Therefore, using values less than 12 will lead to false negative hits. PhiX is a small phage of enterobacteria whose DNA is often spiked-in during Illumina sequencing to determine error rates in datasets and to increase complexity in low-complexity samples amplicon, chipseq etc to help in cluster identification.

If it remains in the sequenced data, it has to be seen as a contaminant for projects working with organisms which should not contain the PhiX phage. For genomes however, MIRA currently is cautious and will not filter these reads by default. Default is is dependent on --job switch: This is a quality ensuring move which improves assembly of ultra-high coverage contigs by cleaning out very likely, low frequency sequence dependent sequencing errors which passed all previous filters.

The drawback is that very rare transcripts with an occurrence less than the given value will also be masked out.

However, RNASeq gives so much data that even the rarest transcripts should normally have more reads than the default setting.

Options that control the behaviour of the initial fast all-against-all read comparison algorithm. Matches found here will be confirmed later in the alignment phase. The new SKIM3 algorithm that is in place since version 2. Number of threads used in SKIM, default is 2.

A few parts of SKIM are non-threaded, so the speedup is not exactly linear, but it should be very close. Although the main data structures are shared between the threads, there's some additional memory needed for each thread. You usually will not want to touch the default, except for very special application cases where you do not want MIRA to use reverse complement sequences at all.

Controls the number of consecutive bases n which are used as a word hash. The higher the value, the faster the search. The lower the value, the more weak matches are found. Values below 10 are not recommended. Defaults are dependent on "--job" switch. This is a parameter controlling the stepping increment s with which hashes are generated. Default is dependent of the sequencing technology used and assembly quality wished.

Controls the relative percentage of exact word matches in an approximate overlap that has to be reached to accept this overlap as possible match. Increasing this number will decrease the number of possible alignments that have to be checked by Smith-Waterman later on in the assembly, but it also might lead to the rejection of weaker overlaps i. Controls the maximum number of possible hits one read can maximally transport to the graph edge reduction phase. If more potential hits are found, only the best ones are taken.

It still is if you run out of memory in the graph edge reduction phase. Try then to lower it to , or even As the assembly increases in passes [-AS: So the accuracy of the assembly should only suffer when lowering this number too much.

Default is currently 3. Takes effect only in mapping assemblies. You will want to set this option to yes whenever your reference sequence contains more complex or numerous repeats and your data has SNPs in those areas.

If the number of reads identified as megahubs exceeds the allowed ratio, MIRA will abort. This is a fail-safe parameter to avoid assemblies where things look fishy. Eukaryotes are likely to contain megahubs if filtering is [-HS: EST project however, especially from non-normalised libraries, will very probably contain megahubs. In this case, you might want to think about masking, see [-HS: Has no influence on the quality of the assembly, only on the maximum memory size needed during the skimming.

The default value is equivalent to approximately MB. On the other hand, increasing the default value chosen will not result in speed improvements that are really noticeable. Default is , when Solexa sequences are used. Maximum memory used in MiB during the reduction of skim hits. As soon as assembling something bigger than 20 megabases, you should increase it to or equivalent to 2 or 4 GiB of memory.

Hash statistics sometimes also called kmer statistics in literature or other software packages allows to quickly assess reads from a coverage point of view without actually assembling the reads. MIRA uses this as a quick pre-assembly evaluation to find and tag reads which are from repetitive and non-repetitive parts of a project.

The length of the hash the kmer size is defined via [-SK: A more in-depth description on hash statistics is given in the sections Introduction to 'masking' and How does 'nasty repeat' masking work?

During hash statistics analysis, MIRA will estimate how repetitive parts of reads are. Parts which are occurring less than [-HS: Parts which are occurring more than [-HS: Default is dependent on --job type: The threshold X from which on subsequences are considered nasty is set by [-HS: This option is extremely useful for assembly of larger projects fungi-size with a high percentage of repeats. Or in non-normalised EST projects, to get at least the sequence of overrepresented transcripts assembled even if the coverage values then cannot be interpreted as expression values anymore.

Normally it's high around for genome assemblies, but much lower 20 or less for EST assemblies. Sets the ratio from which on subsequences are considered nasty and hidden from the hash statistics overlapper with a MNRr tag. A value of 10 means: Closely related to the [-HS: Note that this parameter will take precedence over [-HS: A value of 0 de-activates this parameter. This tag has an active masking function in MIRA and the fast all-against-all overlap searcher SKIM will then completely ignore the tagged subsequences of reads.

There's one drawback though: Reads completely covered by the MNRr tag will therefore land in the debris file as no overlap will be found. When set to yes , MIRA will apply a modified digital normalisation step to the reads, effectively decreasing the coverage of a given repetitive stretch down to a minimum needed to correctly represent one copy of the repeat.

A value of 0 means "switched off". The align options control the behaviour of the Smith-Waterman alignment routines. Only read pairs which are confirmed here may be included into contigs. Affects both the checking of possible alignments found by SKIM as well as the phase when reads are integrated into a contig. Every option in this section can be set individually for every sequencing technology, giving a very fine grained control on how reads are aligned for each technology.

The banded Smith-Waterman alignment uses this percentage number to compute the bandwidth it has to use when computing the alignment matrix. Minimum bandwidth in bases to each side. Maximum bandwidth in bases to each side. Minimum number of overlapping bases needed in an alignment of two sequences to be accepted.

Describes the minimum score of an overlap to be taken into account for assembly. Take a bigger score to weed out a number of chance matches, a lower score to perhaps find the single short alignment that might join two contigs together at the expense of computing time and memory. Increasing this number will save memory, but one might loose possible alignments.

I propose a maximum of 80 here. When running in mapping mode, this defines the maximum number of mismatches and gaps a read may have compared to the reference to be allowed to map. The default value of -1 lets MIRA choose this value automatically.

Defines whether or not to increase penalties applied to alignments containing long gaps. Setting this to 'yes' might help in projects with frequent repeats. On the other hand, it is definitively disturbing when assembling very long reads containing multiple long indels in the called base sequence When in doubt, set it to yes for EST projects and de-novo genome assembly, set it to no for assembly of closely related strains assembly against a backbone. When set to no , it is recommended to have [-CO: Defines an extra penalty applied to 'long' gaps.

There are these are predefined levels: It's even a tick harsher that the 'high' level. Also, usage of this parameter is probably a good thing if the repeat marker of the contig is set to not mark on gap bases [-CO: This is generally the case for data. Defines the maximum extra penalty in percent applied to 'long' gaps. Contigs will have this string prepended to their names. Lower values mean stricter checking.

This value is doubled should a read be entered that has a template partner a read pair at the right distance. One of the most important switches in MIRA: Only takes effect when [-CO: If set to yes , MIRA will not use the repeat resolving algorithm during build time and therefore will not be able to take advantage of this , but only before saving results to disk. Usually, MIRA will mark bases that differentiate between repeats when a conflict occurs between reads that belong to one strain.

If the conflict occurs between reads belonging to different strains, they are marked as SNP. However, if this switch is set to yes , conflict within a strain are also marked as SNP. This switch is mainly used in assemblies of ESTs, it should not be set for genomic assembly. A group is defined by the reads carrying the same nucleotide for a given position, i.

Setting this to a low number increases sensitivity, but might produce a few false positives, resulting in reads being thrown out of contigs because of falsely identified possible repeat markers or wrongly recognised as SNP. Takes only effect when [-CO: This defines the minimum quality of neighbouring bases that a base must have for being taken into consideration during the decision whether column base mismatches are relevant or not.

This defines the minimum quality of a group of bases to be taken into account as potential repeat marker. The lower the number, the more sensitive you get, but lowering below 25 is not recommended as a lot of wrongly called bases can have a quality approaching this value and you'd end up with a lot of false positives. The higher the overall coverage of your project, the better, and the higher you can set this number.

A value of 35 will probably remove most false positives, a value of 40 will probably never show false positives Using the end of sequences of Sanger type shotgun sequencing is always a bit risky, as wrongly called bases tend to crowd there or some sequencing vector relics hang around. It is even more risky to use these stretches for detecting possible repeats, so one can define an exclusion area where the bases are not used when determining whether a mismatch is due to repeats or not.

Setting this parameter will automatically do this. Determines whether columns containing gap bases indels are also tagged. Takes effect only when [-CO: Determines whether multiple columns containing gap bases indels are also tagged. Determines whether both for tagging columns containing gap bases, both strands. Setting this to no is not recommended except when working in desperately low coverage situations. Default is no for all sequencing types. All other things being equal like quality of the possible consensus base and other things , MIRA will choose a base by either looking for a majority vote or, if that also is not clear, by preferring gaps over T over G over C over finally A.

MIRA makes a considerable effort to deduce the right base at each position of an assembly. Only when cases begin to be borderline it will use a IUPAC code to make you aware of potential problems. You might want to set this parameter to yes in the following cases: Default is yes for all Solexas when in a mapping assembly, else it's no.

Can only be used in mapping assemblies. This feature hugely reduces the number of Solexa reads and makes assembly results with Solexa data small enough to be handled by current finishing programs gap4, consed, others on normal workstations. Default is 0 for all Solexas when in a mapping assembly. Takes only effect in mapping assemblies if [-CO: Defines how many "errors" i.

Useful only when one does not need SNP information from an assembly but wants to concentrate either on coverage data or on paired-end information at contig ends.

Default is -1 for all Solexas when in a mapping assembly. Instead of a fixed value, one can also use MIRA will then automatically not merge reads if the distance from the contig end is within the maximum size of the template insert size of the sequencing library for that read either given via [-GE: This feature allows to use the data reduction from [-CO: General options for controlling the integrated automatic editor. The editors generally make a good job cleaning up alignments from typical sequencing errors like like base overcalls etc.

However, they may prove tricky in certain situations:. Usage must be carefully weighed. However, this can make post processing of assembly results pretty difficult with some formats like ACE, where the format itself contains no way to specify certain edits like deletion.

When set to yes, MIRA will use built-in versions of own automatic contig editors see parameters below to improve alignments. Default is yes for all sequencing technologies, but only takes effect if [-ED: When set to yes, MIRA uses the alignment information of a complete contig at places with sequencing errors which lead to unique kmers and correct the error according to the alignment.

This is an extremely conservative yet very effective editing strategy and can therefore be kept always activated. Default is yes for and Ion Torrent, but only takes effect if [-ED: When set to yes, MIRA use the alignment information of a complete contig at places with potential homopolymer sequencing errors and correct the error according to the alignment. This editor should be switched on only for sequencing technologies with known homopolymer sequencing problems.

When set to yes, MIRA will use built-in versions of the "EdIt" automatic contig editor see parameters below to correct sequencing errors in Sanger reads. EdIt will try to resolve discrepancies in the contig by performing trace signal analysis and correct even hard to resolve errors. Only for Sanger data. If set to yes, the automatic editor will not take error hypotheses with a low probability into account, even if all the requirements to make an edit are fulfilled. The higher this value, the more strict the automatic editor will apply its internal rule set.

Going below 40 is not recommended. This switch tells MIRA that you know what you do in some situations and force it not to stop when it thinks something is really wrong, but simply continue. This parameter has absolutely no influence whatsoever on the assembly process of MIRA. MIRA uses coverage information of an assembly project to find out about potentially repetitive areas in reads and thus, a genome.

To calculate statistics which are reflecting the approximate truth regarding the average coverage of a genome, the "large contig size for stats" value of [-MI: This reflects two facts: On the other hand, reads which could not be placed into contigs maybe due to a sequencing technology dependent motif error often enough form small contigs with extremely low coverage. It should be clear that one does not want any of the above when calculating average coverage statistics and having this cutoff discards small contigs which tend to muddy the picture.

If in doubt, don't touch this parameter. Parameters which let MIRA warn you about unusual things or potential problems. The flags in this parameter section come in three flavours: You have been warned. MIRA will check for duplicate read names after loading.

Duplicate read names usually hint to a serious problem with your input and should really, really be fixed. You can choose to ignore this error by switching off this flag, but this will almost certainly lead to problems with result files ACE and CAF for sure, maybe also SAM and probably to other unexpected effects.

MIRA will check read template naming after loading. Problems in read template naming point to problems with read names or to broken template information. You should try to find the cause of the problem instead of ignoring this error message.

MIRA will check whether the length of the names of your reads surpass the given number of characters see [-NW: While MIRA and many other programs have no problem with long read names, some older programs have restrictions concerning the length of the read name. This should be a warning only, but as a couple of people were bitten by this, the default behaviour of MIRA is to stop when it sees that potential problem. On the other hand, you also can ignore this potential problem and force MIRA to continue by using the parameter: This defines the effective check length for [-NW: In genome de-novo assemblies, MIRA will perform checks early in the assembly process whether the average coverage to be expected exceeds a given value see [-NW: With todays' sequencing technologies especially Illumina, but also Ion Torrent and , many people simply take everything they get and throw it into an assembly.

Which, in the case of Illumina and Ion, can mean they try to assemble their organism with a coverage of x, x and more I've seen trials with more than x. The first reason is that, usually, one does not sequence a single cell but a population of cells. If this population is not clonal i. Which, of course, will be wrong and I am pretty sure you do not want that.

The second and way more important reason is that none of the current sequencing technologies is completely error free. Even more problematic, they contain both random and non-random sequencing errors. Especially the latter can become a big hurdle if these non-random errors are so prevalent that they suddenly appear to be valid sequence to an assembler.

This in turn leads to false repeat detection, hence possibly contig breaks or even wrong consensus sequence. You don't want that, do you? The last reason is that overlap based assemblers like MIRA is need exponentially more time and memory when the coverage increases.

So keeping the coverage comparatively low helps you there. Default is 80 for de-novo assemblies, in mapping assemblies it is for Ion Torrent and for Illumina data might change in future. This defines the effective coverage to check for in [-NW: Default is an empty string. This option is particularly useful for systems which have solid state disks SSDs and some very fast disk subsystems which can be used for temporary files.

Or in projects where the input and output files reside on a NFS mounted directory current working dir , to put the tmp directory somewhere outside the NFS see also: Things you should not do. Options for controlling which results to write to which type of files. Additionally, a few options allow output customisation of textual alignments in text and HTML files. There are 3 types of results: One probably needs only the results. Temporary and extra temporary results are written while building different stages of a contig and are given as convenience for trying to find out why MIRA set some RMBs or disassembled some contigs.

Output can be generated in these formats: Controls whether 'unimportant' singlets are written to the result files. Controls whether singlets which have certain tags see below are written to the result files, even if [-OUT: To give the possibility to human finishers to trace back the decision, these singlets can be written to result files.

Removes log and temporary files once they should not be needed anymore during the assembly process. Removes the complete tmp directory at the end of the assembly process. Some logs and temporary files contain useful information that you may want to analyse though, therefore the default of MIRA is not to delete it.

However, due to a bug in consed, consed cannot correctly load tags set by MIRA. There is a workaround: Use the script like this:. When producing an output in text format [-OUT: Default is a blank. It may happen that a MIRA run is interrupted - sometimes rather harshly - due to events more or less outside your control like, e.

This may be less of a problem when assembling or mapping small data sets with run times between a couple of minutes up to a few hours, but becomes a nuisance for larger data sets like in small eukaryotes or RNASeq samples where the run time is measured in days.

For cases like these, MIRA has a resume functionality: Starting MIRA in resume mode is pretty easy: This directory contains up to four sub-directories:. They provide statistics as well as, e.

It can be safely removed after an assembly as there may be easily a few GB of data in there that are not normally not needed anymore. In case of problems: I will get in touch with you for additional information that might possibly be present in the tmp directory.

Type gap4da is a directory containing experiment files and a file of filenames called 'fofn' , all other types are files. Please note that they come in two flavours: Unpadded versions have these gaps removed. Padded versions have an additional postfix. This file contains basic information about the assembly. MIRA will split the information in two parts: For more information on how to interpret this file, please consult the chapter on "Results" of the MIRA documentation manual.

This file contains information which reads have been assembled into which contigs or singlets. This file contains statistics about the contigs themselves, their length, average consensus quality, number of reads, maximum and average coverage, average read length, number of A, C, G, T, N, X and gaps in consensus. This file contains information about the tags and their position that are present in the consensus of a contig.

For de-novo assemblies, this file contains the name of the contigs which pass the adaptable 'large contig' criterion. This file permits a quick analysis of the repetitiveness of different parts of reads in a project. A list containing the names of those reads that have been sorted out of the assembly before any processing started only due to the fact that they were too short. This file contains information about the tags and their position that are present in each read.

The read positions are given relative to the forward direction of the sequence i. These files collect warning messages MIRA dumped out throughout the assembly process.

There are three warning files representing different levels of criticality: These files may be empty, meaning that no warning of the corresponding level was printed. It is strongly suggested to have a look at least at critical warnings during and after an assembly run.

A list of sequences that have been found to be invalid due to various reasons given in the output of the assembler.

This old assembly file format used mainly by phrap and consed. Using consed, you will need to load projects with -nophd to view them. The solution to that is easy: If you don't have consed, you might want to try clview http: Standard experiment files used in genome sequencing. Correct EXP files are expected. Especially the ID record containing the id of the reading and the LN record containing the name of the corresponding trace file should be correctly set. A simple format for sequence data, see http: An often used extension of that format is used to also store quality values in a similar fashion, these files have a.

The difference is that the padded version still contains the gap pad character an asterisk at positions in the consensus where some of the reads apparently had some more bases than others but where the consensus routines decided that to treat them as artifacts. The unpadded version has the gaps removed. MIRA is able to read and write this format but only for viruses or bacteria for using sequences as backbones in an assembly.

Features of the GenBank format are also transferred automatically to Staden compatible tags. General feature format used to describe sequences and features on these sequences.

MIRA is able to read and write this format. Projects written in HTML format can be viewed directly with any table capable browser. Display is even better if the browser knows style sheets CSS.