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Bowtie 2 is an ultrafast and memory-efficient tool 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 optionen trading card game pokemon online freezer-codes 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 optionen trading card game pokemon online freezer-codes 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. Optionen trading card game pokemon online freezer-codes 1 does not. In Bowtie 2 all alignments lie along a continuous spectrum of 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 optionen trading card game pokemon online freezer-codes 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 optionen trading card game pokemon online freezer-codes 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 optionen trading card game pokemon online freezer-codes 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 most 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 optionen trading card game pokemon online freezer-codes, including bowtie2bowtie2-align-sbowtie2-align-lbowtie2-buildoptionen trading card game pokemon online freezer-codesbowtie2-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 the —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 Optionen trading card game pokemon online freezer-codes 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 open, -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 how 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 optionen trading card game pokemon online freezer-codes 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 optionen trading card game pokemon online freezer-codes, 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 optionen trading card game pokemon online freezer-codes 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 fields 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 sequenced.
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 optionen trading card game pokemon online freezer-codes uniquely. This is a conservative threshold, but this is often desirable when seeking structural variants.
By default, Bowtie 2 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 optionen trading card game pokemon online freezer-codes, 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 optionen trading card game pokemon online freezer-codes and the other mate in the pair aligned to the Crick strand or, equivalently, if the reverse complement of the other 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 fields 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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