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Bowtie 2 is an ultrafast and memory-efficient handel mit binare optionen bei 5 minuten template for aligning sequencing reads to long reference sequences.

It is particularly good at aligning reads of about 50 up to s or 1,s of characters to relatively long e.

Bowtie 2 supports gapped, local, and paired-end alignment modes. Multiple processors can be used simultaneously to achieve greater alignment speed. If you use Bowtie 2 for your published research, please cite the Bowtie paper. Bowtie 1 was released in and was geared toward aligning the relatively short sequencing reads up to nucleotides prevalent at the time. Since then, technology has improved both sequencing throughput more nucleotides produced per sequencer per day and read length more nucleotides per read.

For reads longer than about 50 bp Bowtie 2 is generally faster, more sensitive, and uses less memory than Bowtie 1. For relatively short reads e. Bowtie 2 supports gapped alignment with affine gap penalties. Number of gaps and gap lengths are not restricted, except by way of the configurable scoring scheme. Bowtie 1 finds just ungapped alignments. Bowtie 2 also supports end-to-end alignment which, like Bowtie 1, requires that the read align entirely.

There is no upper limit on read length in Bowtie 2. Bowtie 1 had an upper limit of around bp. Bowtie 2 allows alignments to overlap ambiguous characters e. N s in the reference. Bowtie 1 does not. In Bowtie 2 all alignments lie along a continuous spectrum handel mit binare optionen bei 5 minuten template alignment scores where the scoring schemesimilar to Needleman-Wunsch and Smith-Waterman.

Bowtie 2 reports a spectrum of mapping qualities, in contrast for Bowtie 1 which reports either 0 or high. Bowtie 2 works best when aligning to large genomes, though it supports arbitrarily small reference sequences e. It handles very long reads i. If your goal is to align two very large sequences e. If your goal is very sensitive alignment to a relatively short reference sequence e. These tools can be extremely slow when the reference genome is long, but are often adequate when the reference is short.

Download Bowtie 2 sources and binaries from the Download section of the Sourceforge site. If you plan to compile Bowtie 2 yourself, make sure to get the source package, i. It should be possible to build Bowtie 2 on most vanilla Linux installations or on a Mac installation with Xcode installed. In order to simplify the MinGW setup it might be worth investigating popular MinGW personal builds since these are coming already prepared with most of the toolchains needed.

First, download the source package from the sourceforge site. Unzip the file, change to the unzipped directory, and build the Bowtie 2 tools by running GNU make usually with the command makebut sometimes with gmake with no arguments. Bowtie 2 is using the multithreading software model in order to speed up execution times on SMP architectures where this is possible. Although it is possible to use pthread library on Windows, a non-POSIX platform, due to performance reasons Bowtie 2 will try to use Windows native multithreading if possible.

TBB comes installed by default on many popular Linux distros. Please note, packages built without TBB will have -legacy appended to the name. By adding your new Bowtie 2 directory to your PATH environment variableyou ensure that whenever you run bowtie2bowtie2-build or bowtie2-inspect from the command line, you will get the version you just installed without having to specify the entire path. This is recommended for handel mit binare optionen bei 5 minuten template users.

If you would like to install Bowtie 2 by copying the Bowtie 2 executable files to an existing directory in your PATHmake sure that you copy all the executables, including bowtie2bowtie2-align-sbowtie2-align-lbowtie2-buildbowtie2-build-sbowtie2-build-lbowtie2-inspectbowtie2-inspect-s and bowtie2-inspect-l. We use alignment to make an educated guess as to where a read originated with respect to the reference genome. By default, Bowtie 2 performs end-to-end read alignment.

That is, it searches for alignments involving all of the read characters. When handel mit binare optionen bei 5 minuten template —local option is specified, Bowtie 2 performs local read alignment. Such an alignment can be produced by Bowtie 2 in either end-to-end mode or in local mode. This sort of alignment can be produced by Bowtie 2 only in local mode. An alignment score quantifies how similar the read sequence is to the reference sequence aligned to. The higher the score, the more similar they are.

A score is calculated by subtracting penalties for each difference mismatch, gap, etc. The scores can be configured with the --ma match bonus--mp mismatch penalty--np penalty for having an N in either the read or the reference--rdg affine read gap penalty and --rfg affine reference gap penalty options.

A mismatched base at a high-quality position in the read receives a penalty of -6 by default. A length-2 read gap receives a penalty of by default -5 for the gap handel mit binare optionen bei 5 minuten template, -3 for the first extension, -3 for the second extension.

The best possible alignment score in end-to-end mode is 0, which happens when there are no differences between the read and the reference.

The best possible score in local mode equals the match bonus times the length of the read. This happens when there are no differences between the read and the reference. The threshold is configurable and is expressed as a function of the read length. In end-to-end alignment mode, the default minimum score threshold is This can be configured with the --score-min option. For details on handel mit binare optionen bei 5 minuten template to set options like --score-min that correspond to functions, see the section on setting function options.

The aligner cannot always assign a read to its point of origin with high confidence. For instance, a read that originated inside a repeat element might align equally well to many occurrences of the element throughout the genome, leaving the aligner with no basis for preferring one over the others. Aligners characterize their degree of confidence in the point of origin by reporting a mapping quality: Accurate mapping qualities are useful for downstream tools like variant callers.

For instance, a variant caller might choose to ignore evidence from alignments with mapping quality less than, say, A mapping quality of 10 or less indicates that there is at least a 1 in 10 chance that the read truly originated elsewhere. Pairs come with a prior expectation about a the relative orientation of the mates, and b the distance separating them on the original DNA molecule.

Exactly what expectations hold for a given dataset depends on the lab procedures used to generate the data. Also, this protocol yields pairs where the expected genomic distance from end to end is about base pairs. Pairs are often stored in a pair of files, one file containing the mate 1s and the other containing the mates 2s.

The first mate in the file for mate 1 forms a pair with the first mate in the file for mate 2, the second with the second, and so on. When aligning pairs with Bowtie 2, specify the file with the mate 1s mates using the -1 argument and the file with the mate 2s using the -2 argument. This causes Bowtie 2 to take the paired nature of the reads into account when aligning them.

When Bowtie 2 prints a SAM alignment for a pair, it prints two records i. The first record describes the alignment for mate 1 and the second record describes the alignment for mate 2.

In both records, some of the fields of the SAM record describe various properties of the alignment; for instance, the 7th and 8th handel mit binare optionen bei 5 minuten template RNEXT and PNEXT respectively indicate the reference name and position where the other mate aligned, and the 9th field indicates the inferred length of the DNA fragment from which the two mates were handel mit binare optionen bei 5 minuten template.

See the SAM specification for more details regarding these fields. If both mates have unique alignments, but the alignments do not match paired-end expectations i. Discordant alignments may be of particular interest, for instance, when seeking structural variants. The expected relative orientation of the mates is set using the --ff--fror --rf options. Note that setting -I and -X far apart makes Bowtie 2 slower.

See documentation for -I and -X. To declare that a pair aligns discordantly, Bowtie 2 requires that both mates align uniquely. This is a conservative threshold, but this is often desirable handel mit binare optionen bei 5 minuten template seeking structural variants.

By default, Bowtie handel mit binare optionen bei 5 minuten template searches for both concordant and discordant alignments, though searching for discordant alignments can be disabled with the --no-discordant option. If Bowtie 2 cannot find a paired-end alignment for a pair, by default it will go on to look for unpaired alignments for the constituent mates.

Bowtie 2 runs a little faster in --no-mixed mode, but will only consider alignment status of pairs per se, not individual mates. The first least significant bit 1 in decimal, 0x1 in hexadecimal is set if the read is part of a pair. The second bit 2 in decimal, 0x2 in hexadecimal is set if the read is part of a pair that aligned in a paired-end fashion.

The fourth bit 8 in decimal, 0x8 in hexadecimal is set if the read is part of a pair and the other mate in the pair had at least one valid alignment. The sixth bit 32 in decimal, 0x20 in hexadecimal is set if the read is part of a pair and the other mate in the pair aligned to the Crick strand or, equivalently, if the reverse complement of the handel mit binare optionen bei 5 minuten template mate aligned to the Watson strand.

The seventh bit 64 in decimal, 0x40 in hexadecimal is set if the read is mate 1 in a pair. The eighth bit in decimal, 0x80 in hexadecimal is set if the read is mate 2 in a pair.

The last several handel mit binare optionen bei 5 minuten template of each SAM record usually contain SAM optional fields, which are simply tab-separated strings conveying additional information about the reads and alignments. A SAM optional field is formatted like this: The fragment and read lengths might be such that alignments for the two mates from a pair overlap each other. By default, dovetailing is considered inconsistent with concordant alignment. These defaults can be overridden.

Setting --no-overlap causes Bowtie 2 to consider overlapping mates as non-concordant. Setting --no-contain causes Bowtie 2 to consider cases where one mate alignment contains the other as non-concordant. Setting --dovetail causes Bowtie 2 to consider cases where the mate alignments dovetail as concordant. The reporting mode governs how many alignments Bowtie 2 looks for, and how to report them.

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This is a lot more expensive, from a processing-overhead perspective, than your standard read-a-file-off-the-filesystem server arrangement. For convenience, Django offers different levels of cache granularity: You can cache the output of specific views, you can cache only the pieces that are difficult to produce, or you can cache your entire site.

The Cache Framework design philosophy explains a few of the design decisions of the framework. The cache system requires a small amount of setup. Namely, you have to tell it where your cached data should live — whether in a database, on the filesystem or directly in memory.

The fastest, most efficient type of cache supported natively by Django, Memcached is an entirely memory-based cache server, originally developed to handle high loads at LiveJournal.

It is used by sites such as Facebook and Wikipedia to reduce database access and dramatically increase site performance. Memcached runs as a daemon and is allotted a specified amount of RAM. All it does is provide a fast interface for adding, retrieving and deleting data in the cache. There are several Python Memcached bindings available; the two most common are python-memcached and pylibmc. In this example, Memcached is running on localhost When using the pylibmc binding, do not include the unix: One excellent feature of Memcached is its ability to share a cache over multiple servers.

This means you can run Memcached daemons on multiple machines, and the program will treat the group of machines as a single cache, without the need to duplicate cache values on each machine. In this example, the cache is shared over Memcached instances running on IP address In the following example, the cache is shared over Memcached instances running on the IP addresses A final point about Memcached is that memory-based caching has a disadvantage: Django can store its cached data in your database.

If you are using multiple database caches, createcachetable creates one table for each cache. It will only create missing tables. To print the SQL that would be run, rather than run it, use the createcachetable --dry-run option. The file-based backend serializes and stores each cache value as a separate file. The directory path should be absolute — that is, it should start at the root of your filesystem. Make sure the directory pointed-to by this setting exists and is readable and writable by the system user under which your Web server runs.

This is the default cache if another is not specified in your settings file. This cache is per-process see below and thread-safe. If you only have one locmem cache, you can omit the LOCATION ; however, if you have more than one local memory cache, you will need to assign a name to at least one of them in order to keep them separate.

Note that each process will have its own private cache instance, which means no cross-process caching is possible. While Django includes support for a number of cache backends out-of-the-box, sometimes you might want to use a customized cache backend. Each cache backend can be given additional arguments to control caching behavior. Valid arguments are as follows:. The default timeout, in seconds, to use for the cache. This argument defaults to seconds 5 minutes.

Any options that should be passed to the cache backend. The list of valid options will vary with each backend, and cache backends backed by a third-party library will pass their options directly to the underlying cache library. Cache backends that implement their own culling strategy i. The maximum number of entries allowed in the cache before old values are deleted.

This argument defaults to This argument should be an integer and defaults to 3. On some backends database in particular this makes culling much faster at the expense of more cache misses. Memcached backends pass the contents of OPTIONS as keyword arguments to the client constructors, allowing for more advanced control of client behavior. For example usage, see below. A string that will be automatically included prepended by default to all cache keys used by the Django server.

See the cache documentation for more information. The default version number for cache keys generated by the Django server. In this example, a filesystem backend is being configured with a timeout of 60 seconds, and a maximum capacity of items:.

Once the cache is set up, the simplest way to use caching is to cache your entire site. Responses to requests for the same URL with different query parameters are considered to be unique pages and are cached separately. In older versions, the Last-Modified header was also set.

See Middleware for more on middleware. If a view sets its own cache expiry time i. Using the decorators in django. See the using other headers section for more on these decorators.

This allows you to easily cache multilingual sites without having to create the cache key yourself. A more granular way to use the caching framework is by caching the output of individual views. The per-view cache, like the per-site cache, is keyed off of the URL. But once a particular URL e. By default, the default cache will be used, but you can specify any cache you want:.

You can also override the cache prefix on a per-view basis. It can be used like this:. This approach couples your view to the cache system, which is not ideal for several reasons. For instance, you might want to reuse the view functions on another, cache-less site, or you might want to distribute the views to people who might want to use them without being cached.

The solution to these problems is to specify the per-view cache in the URLconf rather than next to the view functions themselves. Doing so is easy: It takes at least two arguments: The fragment is cached forever if timeout is None. The name will be taken as is, do not use a variable. Sometimes you might want to cache multiple copies of a fragment depending on some dynamic data that appears inside the fragment.

For example, you might want a separate cached copy of the sidebar used in the previous example for every user of your site. For the cache template tag you could use one of the translation-specific variables available in templates to achieve the same result:. The cache timeout can be a template variable, as long as the template variable resolves to an integer value. This feature is useful in avoiding repetition in templates. You can set the timeout in a variable, in one place, and just reuse that value.

If no such cache exists, it will fall back to using the default cache. You may select an alternate cache backend to use with the using keyword argument, which must be the last argument to the tag.

This function can be useful for invalidating or overwriting a cached item, for example:. Perhaps, for instance, your site includes a view whose results depend on several expensive queries, the results of which change at different intervals. For cases like this, Django exposes a simple, low-level cache API. You can use this API to store objects in the cache with any level of granularity you like.

You can cache any Python object that can be pickled safely: Most common Python objects can be pickled; refer to the Python documentation for more information about pickling. Repeated requests for the same alias in the same thread will return the same object. If the named key does not exist, InvalidCacheBackendError will be raised. To provide thread-safety, a different instance of the cache backend will be returned for each thread.

As a shortcut, the default cache is available as django. This object is equivalent to caches['default']. The basic interface is set key, value, timeout and get key:.

The timeout argument is optional and defaults to the timeout argument of the appropriate backend in the CACHES setting explained above. Passing in None for timeout will cache the value forever. It takes the same parameters as set , but it will not attempt to update the cache if the key specified is already present:.

If you need to know whether add stored a value in the cache, you can check the return value. It will return True if the value was stored, False otherwise.

It takes the same parameters as get but the default is set as the new cache value for that key, rather than simply returned:. You can delete keys explicitly with delete. This is an easy way of clearing the cache for a particular object:. Finally, if you want to delete all the keys in the cache, use cache.

Be careful with this; clear will remove everything from the cache, not just the keys set by your application. You can also increment or decrement a key that already exists using the incr or decr methods, respectively.