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bowtie2 aligner
bowtie2-build indexer
bowtie2-inspect index inspector
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 100s or 1,000s of characters to relatively long (e.g. mammalian) genomes. Bowtie 2 indexes the genome with an FM Index (based on the Burrows-Wheeler Transform or BWT) to keep its memory footprint small: for the human genome, its memory footprint is typically around 2.3 GB. Bowtie 2 supports gapped, local, and paired-end alignment modes. Multiple processors can be used simultaneously to achieve greater alignment speed. Bowtie 2 outputs alignments in SAM format, enabling interoperation with a large number of other tools (e.g. SAMtools, GATK) that use SAM. Bowtie 2 is distributed under the GPLv3 license, and it runs on the command line under Windows, Mac OS X and Linux.
Bowtie 2 is often the first step in pipelines for comparative genomics, including for variation calling, ChIP-seq, RNA-seq, BS-seq. Bowtie 2 and Bowtie (also called "Bowtie 1" here) are also tightly integrated into some tools, including TopHat: a fast splice junction mapper for RNA-seq reads, Cufflinks: a tool for transcriptome assembly and isoform quantitiation from RNA-seq reads, Crossbow: a cloud-enabled software tool for analyzing reseuqncing data, and Myrna: a cloud-enabled software tool for aligning RNA-seq reads and measuring differential gene expression.
If you use Bowtie 2 for your published research, please cite the Bowtie paper. Thank you!
Bowtie 1 was released in 2009 and was geared toward aligning the relatively short sequencing reads (up to 25-50 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).
The chief differences between Bowtie 1 and Bowtie 2 are:
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.g. less than 50 bp) Bowtie 1 is sometimes faster and/or more sensitive.
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 supports local alignment, which doesn't require reads to align end-to-end. Local alignments might be "trimmed" ("soft clipped") at one or both extremes in a way that optimizes alignment score. 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 1000 bp.
Bowtie 2 allows alignments to overlap ambiguous characters (e.g.
Ns) in the reference. Bowtie 1 did not.
Bowtie 2 does away with Bowtie 1's notion of alignment "stratum", and its distinction between "Maq-like" and "end-to-end" modes. In Bowtie 2 all alignments lie along a continuous spectrum of alignment scores where the scoring scheme, similar to Needleman-Wunsch and Smith-Waterman.
Bowtie 2's paired-end alignment is more flexible. E.g. for pairs that do not align in a paired fashion, Bowtie 2 attempts to find unpaired alignments for each mate.
Bowtie 2 does not align colorspace reads.
Bowtie 2 is not a "drop-in" replacement for Bowtie 1. Bowtie 2's command-line arguments and genome index format are both different from Bowtie 1's.
Bowtie 1 and Bowtie 2 are not general-purpose alignment tools like MUMmer, BLAST or Vmatch. Bowtie 2 works best when aligning to large genomes, though it supports arbitrarily small reference sequences (e.g. amplicons). It handles very long reads (i.e. upwards of 10s or 100s of kilobases), but it is optimized for the read lengths and error modes yielded by recent sequencers, such as the Illumina HiSeq 2000, Roche 454, and Ion Torrent instruments.
If your goal is to align two very large sequences (e.g. two genomes), consider using MUMmer. If your goal is very sensitive alignment to a relatively short reference sequence (e.g. a bacterial genome), this can be done with Bowtie 2 but you may want to consider using tools like NUCmer, BLAT, or BLAST. These tools can be extremely slow when the reference genome is long, but are often adequate when the reference is short.
Bowtie 2 does not support alignment of colorspace reads. This might be supported in future versions.
We say that Bowtie 2 is in "beta" to convey (a) that it is not as polished as a tool that has been around for a while, and (b) it is still in flux. Features and file formats might still change in the near future, so please do not depend heavily on these remaining static.
Download Bowtie 2 sources and binaries from the Download section of the Sourceforge site. Binaries are available for Intel architectures (
i386 and
x86_64) running Linux, and Mac OS X. A 32-bit version is available for Windows. If you plan to compile Bowtie 2 yourself, make sure to get the source package, i.e., the filename that ends in "-src.zip".
Building Bowtie 2 from source requires a GNU-like environment with GCC, GNU Make and other basics. It should be possible to build Bowtie 2 on most vanilla Linux installations or on a Mac installation with Xcode installed. Bowtie 2 can also be built on Windows using Cygwin or MinGW (MinGW recommended). If building with MinGW, first install MinGW and MSYS, the zlib library, and the pthreads library. You may also need the GnuWin32 core and other utilities to drive the build process.
First, download the source package from the sourceforge site. Make sure you're getting the source package; the file downloaded should end in
-src.zip. Unzip the file, change to the unzipped directory, and build the Bowtie 2 tools by running GNU
make (usually with the command
make, but sometimes with
gmake) with no arguments. If building with MinGW, run
make from the MSYS environment.
To support the
-p (multithreading) option, Bowtie 2 needs the
pthreads library. To compile Bowtie 2 without
pthreads (which disables
-p), use
make BOWTIE_PTHREADS=0.
By adding your new Bowtie 2 directory to your PATH environment variable, you ensure that whenever you run
bowtie2-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. To do this, follow your operating system's instructions for adding the directory to your PATH.
bowtie2 aligner
bowtie2 takes a Bowtie 2 index and a set of sequencing read files and outputs a set of alignments in SAM format.
"Alignment" is the process by which we discover how and where the read sequences are similar to the reference sequence. An "alignment" is a result from this process, specifically: an alignment is a way of "lining up" some or all of the characters in the read with some characters from the reference in a way that reveals how they're similar.
We use alignment to make an educated guess as to where a read originated with respect to the reference genome. It's not always possible to determine this with certainty. For instance, if the reference genome contains several long stretches of As (
AAAAAAAAA etc) and the read sequence is a short stretch of As (
AAAAAAA), we cannot know for certain exactly where in the sea of
As the read originated.
By default, Bowtie 2 performs end-to-end read alignment. That is, it searches for alignments involving all of the read characters. This could also be called an "untrimmed" alignment.
When the --local option is specified, Bowtie 2 performs local read alignment. In this mode, Bowtie 2 might "trim" or "clip" some read characters from one or both ends of the alignment if doing so maximizes the alignment score.
The following is an "end-to-end" alignment because it involves all the characters in the read. Such an alignment can be produced by Bowtie 2 in either end-to-end mode or in local mode.
Read: GACTGGGCGATCTCGACTTCG
Reference: GACTGCGATCTCGACATCG
Read: GACTGGGCGATCTCGACTTCG
||||| |||||||||| |||
Reference: GACTG--CGATCTCGACATCG
The following is a "local" alignment because some of the characters at the ends of the read do not participate. In this case, 4 characters are omitted (or "soft trimmed" or "soft clipped") from the beginning and 3 characters are omitted from the end. This sort of alignment can be produced by Bowtie 2 only in local mode.
Read: ACGGTTGCGTTAATCCGCCACG
Reference: TAACTTGCGTTAAATCCGCCTGG
Read: ACGGTTGCGTTAA-TCCGCCACG
||||||||| ||||||
Reference: TAACTTGCGTTAAATCCGCCTGG
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) and, in local alignment mode, adding bonuses for each match.
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 -11 by default (-5 for the gap open, -3 for the first extension, -3 for the second extension). Thus, in end-to-end alignment mode, if the read is 50 bp long and it matches the reference exactly except for one mismatch at a high-quality position and one length-2 read gap, then the overall score is -(6 + 11) = -17.
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.
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 -11 by default (-5 for the gap open, -3 for the first extension, -3 for the second extension). A base that matches receives a bonus of +2 be default. Thus, in local alignment mode, if the read is 50 bp long and it matches the reference exactly except for one mismatch at a high-quality position and one length-2 read gap, then the overall score equals the total bonus, 2 * 49, minus the total penalty, 6 + 11, = 81.
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.
For an alignment to be considered "valid" (i.e. "good enough") by Bowtie 2, it must have an alignment score no less than the minimum score threshold. The threshold is configurable and is expressed as a function of the read length. In end-to-end alignment mode, the default minimum score threhsold is -0.6 + -0.9 * L, where L is the read length. In local alignment mdoe, the default minimum score threshold is 0.66 * L, where L is the read length. This can be configured with the
--score-min option. For details on how to set options like
--score-min that correpond 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: a non-negative integer Q = -10 log10 p, where p is an estimate of the probability that the alignment does not correspond to the read's true point of origin. Mapping quality is sometimes abbreviated MAPQ, and is recorded in the SAM
MAPQ field.
Mapping quality is related to "uniqueness." We say an alignment is unique if it has a much higher alignment score than all the other possible alignments. The bigger the gap between the best alignment's score and the second-best alignment's score, the more unique the best alignment, and the higher its mapping quality should be.
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, 10. A mapping quality of 10 or less indicates that there is at least a 1 in 10 chance that the read truly originated elsewhere.
A "paired-end" or "mate-pair" read consists of pair of mates, called mate 1 and mate 2. 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. For example, a common lab procedure for producing pairs is Illumina's Paired-end Sequencing Assay, which yields pairs with a relative orientation of FR ("forward, reverse") meaning that if mate 1 came from the Watson strand, mate 2 very likely came from the Crick strand and vice versa. Also, this protocol yields pairs where the expected genomic distance from end to end is about 200-500 base pairs.
For simplicity, this manual uses the term "paired-end" to refer to any pair of reads with some expected relative orientation and distance. Depending on the protocol, these might actually be referred to as "paired-end" or "mate-paired." Also, we always refer to the individual sequences making up the pair as "mates."
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.e. two lines of output), one for each mate. 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.
A pair that aligns with the expected relative mate orientation and with the expected range of distances between mates is said to align "concordantly". If both mates have unique alignments, but the alignments do not match paired-end expectations (i.e. the mates aren't in the expcted relative orientation, or aren't within the expected disatance range, or both), the pair is said to align "discordantly". Discordant alignments may be of particular interest, for instance, when seeking structural variants.
The expected relative orientation of the mates is set using the
--fr, or
--rf options. The expected range of inter-mates distances (as measured from the furthest extremes of the mates; also called "outer distance") is set with the
-I and
-X options.
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 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. This is called "mixed mode." To disable mixed mode, set the
--no-mixed option.
Bowtie 2 runs a little faster in
--no-mixed mode, but will only consider alignment status of pairs per se, not individual mates. I.e. in SAM records for pairs, the optional YM:i flag will not appear and MAPQ values calculated for mates may be underestimates in some cases.
The SAM
FLAGS field, the second field in a SAM record, has multiple bits that describe the paired-end nature of the read and alignment. The first (least significant) bit (1 in decimal, 0x1 in hexidecimal) is set if the read is part of a pair. The second bit (2 in decimal, 0x2 in hexidecimal) 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 hexidecimal) 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 hexidecimal) 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 other mate aligned to the Watson strand). The seventh bit (64 in decimal, 0x40 in hexidecimal) is set if the read is mate 1 in a pair. The eighth bit (128 in decimal, 0x80 in hexidecimal) is set if the read is mate 2 in a pair. See the SAM specification for a more detailed description of the
FLAGS field.
The last severeal 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: "XP:i:1" where "XP" is the
TAG, "i" is the
TYPE ("integer" in this case), and "1" is the
VALUE. See the SAM specification for details regarding SAM optional fields.
The following SAM optional fields are sometimes output by Bowtie 2 to convey information about how the pair and its constitutuent mates aligned:
YP:i If present and set to 1, the number of valid, distinct, concordant alignments for the pair is greater than or equal to the ceiling set with the
-M option. If present and set to 0, the number of valid, distinct, concordant alignments is less than the ceiling set with the
-M option. If not present, the alignment is not paired or no
-M ceiling is set.
YM:i If present and set to 1, the number of valid, distinct, unpaired alignments for the mate exceeds the ceiling set with the
-M option. If present and set to 0, the number of valid, distinct, unpaired alignments for the mate is less than the ceiling set with the
-M option. If not present, the read is not part of a pair or no
-M ceiling was set.
The fragment and read lengths might be such that alignments for the two mates from a pair overlap each other. Consider this example:
(For these examples, assume we expect mate 1 to align to the left of mate 2.)
Mate 1: GCAGATTATATGAGTCAGCTACGATATTGTT
Mate 2: TGTTTGGGGTGACACATTACGCGTCTTTGAC
Reference: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGCGTCTTTGAC
It's also possible, though unusual, for one mate alignment to contain the other, as in these examples:
Mate 1: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGC
Mate 2: TGTTTGGGGTGACACATTACGC
Reference: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGCGTCTTTGAC
Mate 1: CAGCTACGATATTGTTTGGGGTGACACATTACGC
Mate 2: CTACGATATTGTTTGGGGTGAC
Reference: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGCGTCTTTGAC
And it's also possible, though unusual, for the mates to "dovetail", with the mates seemingly extending "past" each other as in this example:
Mate 1: GTCAGCTACGATATTGTTTGGGGTGACACATTACGC
Mate 2: TATGAGTCAGCTACGATATTGTTTGGGGTGACACAT
Reference: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGCGTCTTTGAC
In some situations, it's desirable for the aligner to consider all these cases as "concordant" as long as other paired-end constraints are not violated; this is Bowtie 2's default behavior. In other situations, the aligner should consider one or more of these cases to be non-concordant. 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
--no-dovetail causes Bowtie 2 to consider cases where the mate alignments dovetail as non-concordant.
The reporting mode governs how many alignments Bowtie 2 looks for, and how to report them. Bowtie 2 has three distinct reporting modes. The default reporting mode,
-M, is similar to the default reporting mode of many other read alignment tools, including BWA. It is also similar to Bowtie 1's
-M alignment mode.
In general, when we say that a read has an alignment, we mean that it has a valid alignment. When we say that a read has multiple alignments, we mean that it has multiple alignments that are valid and distinct from one another.
Two alignments for the same individual read are "distinct" if they map the same read to different places. Specifically, we say that two alignments are distinct if there are no alignment positions where a particular read offset is aligned opposite a particular reference offset in both alignments with the same orientation. E.g. if the first alignment is in the forward orientation and aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, and the second alignment is also in the forward orientation and also aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, they are not distinct alignments.
Two alignments for the same pair are distinct if either the mate 1s in the two paired-end alignments are distinct or the mate 2s in the two alignments are distinct or both.
-M mode, Bowtie 2 searches for at most N distinct, valid alignments for each read, where N equals the integer specified with the
-M parameter plus one. That is, if
-M 3 is specified, Bowtie 2 will search for at most 4 distinct alignments. Bowtie 2 will stop searching when it can't find more distinct valid alignments, or when it finds N distinct alignments, whichever happens first. It then reports the best alignment found using information about the other alignments to estimate mapping quality and to set SAM optional fields, such as the
AS:i and
XS:i fields.
-M makes Bowtie 2 slower, but increases the likelihood that it will find and report the correct alignment for a read that aligns many places. Bowtie 2 does not "find" alignments in any specific order, so for reads that have more than N distinct, valid alignments, Bowtie 2 does not gaurantee that the alignment reported is the best possible in terms of alignment score.
-k mode, Bowtie 2 searches for up to N distinct, valid alignments for each read, where N equals the integer specified with the
-k parameter. That is, if
-k 2 is specified, Bowtie 2 will search for at most 2 distinct alignments. It reports all alignments found, in descending order by alignment score. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. See the SAM specification for details.
Bowtie 2 does not "find" alignments in any specific order, so for reads that have more than N distinct, valid alignments, Bowtie 2 does not gaurantee that the N alignments reported are the best possible in terms of alignment score.
-a mode is similar to
-k mode except that there is no upper limit on the number of alignments Bowtie 2 should report. Alignments are reported in descending order by alignment score. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. See the SAM specification for details.
To rapidly narrow the number of possible alignments that must be considered, Bowtie 2 begins by extracting substrings ("seeds") from the read and its reverse complement and aligning them in an ungapped fashion with the help of the FM Index. This is "multiseed alignment" and it is similar to what Bowtie 1 does, except Bowtie 1 attempts to align the entire read this way.
This initial step makes Bowtie 2 much faster than it would be without such a filter, but at the expense of missing some valid alignments. For instance, it is possible for a read to have a valid overall alignment but to have no valid seed alignments because each potential seed alignment is interruped by too many mismatches or gaps.
The tradeoff between speed and sensitivity can be adjusted by setting the seed length (
-L), the interval between extracted seeds (
-i), and the number of mismatches permitted per seed (
-N). For more sensitive alignment, set these parameters to (a) make the seeds closer together, (b) make the seeds shorter, and/or (c) allow more mismatches. You can adjust these options one-by-one, though Bowtie 2 comes with some useful combinations of options pre-packaged as "preset options."
Bowtie 2 uses the FM Index to find ungapped alignments for seeds. This step accounts for the bulk of Bowtie 2's memory footprint, as the FM Index itself is typically the largest data structure used. For instance, the memory footprint of the FM Index for the human genome when 0 seed mismatches are permitted (
-N 0) is about 2.3 gigabytes of RAM. When the number of mismatches permitted per seed is greater than 0 (
-N 1 or
-N 2), this necessitates use of the "mirror index" which further increases the memory footprint to about 3.2 gigabytes for the human genome.
-N 2 heuristic
-N 2 is specified, Bowtie 2 allows up to 2 mismatches in seed alignments. In theory, this can lead to "excessive backtracking," where the alignment procedure spends an inordinate amount of time trying various combinations of mismatches. This is disucssed further in the Bowtie 1 paper. To avoid some types of excessive backtracking, Bowtie 2 uses the bi-directional BWT approach first proposed for use in the 2BWT aligner. But as extra insurane against excessive backtracking, Bowtie 2 also employs a heuristic whereby some 2-mismatch alignments won't be found. Specifically, imagine the seed evenly divided into four sections: A, B, C and D. Bowtie 2 could miss seed alignments with mismatches in sections A and C, or in sections B and D. Since the seed-alignment step is a heuristic to begin with, this is not likely a major concern for users.
Non-whitespace characters besides A, C, G or T are considered "ambiguous." N is a common ambiguous character that appears in reference sequences. Bowtie 2 considers all ambiguous characters in the reference (including IUPAC nucleotide codes) to be Ns.
Bowtie 2 allows alignments to overlap ambiguous characters in the reference. An alignment position that contains an ambiguous character in the read, reference, or both, is penalized according to
--n-ceil sets an upper limit on the number of positions that may contain ambiguous reference characters in a valid alignment. The optional field
XN:i reports the number of ambiguous reference characters overlapped by an alignment.
Note that the multiseed heuristic cannot find seed alignments that overlap ambiguous reference characters. For an alignment overlapping an ambiguous reference character to be found, it must have one or more seed alignments that do not overlap ambiguous reference characters.
Bowtie 2 comes with some useful combinations of parameters packaged into shorter "preset" parameters. For example, running Bowtie 2 with the
--very-sensitive option is the same as running with options:
-M 3 -N 0 -L 20 -i S,1,0.50. The preset options that come with Bowtie 2 are designed to cover a wide area of the speed/sensitivity/accuracy tradeoff space, with the presets ending in
fast generally being faster but less sensitive and less accurate, and the presets ending in
sensitive generally being slower but more sensitive and more accurate. See the documentation for the preset options for details.
Some reads are skipped or "filtered out" by Bowtie 2. For example, reads may be filtered out because they are extremely short or have a high proportion of ambiguous nucleotides. Bowtie 2 will still print a SAM record for such a read, but no alignment will be reported and and the
YF:i SAM optional field will be set to indicate the reason the read was filtered.
YF:Z:LN: the read was filtered becuase it had length less than or equal to the number of seed mismatches set with the
-N option.
YF:Z:NS: the read was filtered because it contains a number of ambiguous characters (usually
N or
.) greater than the ceiling specified with
YF:Z:SC: the read was filtered because the read length and the match bonus (set with
--ma) are such that the read can't possibly earn an alignment score greater than or equal to the threshold set with
YF:Z:QC: the read was filtered because it was marked as failing quality control and the user specified the
--qc-filter option. This only happens when the input is in Illumina's QSEQ format (i.e. when
--qseq is specified) and the last (11th) field of the read's QSEQ record contains
If a read could be filtered for more than one reason, the value
YF:Z flag will reflect only one of those reasons.
When Bowtie 2 finishes running, it prints messages summarizing what happened. These messages are printed to the "standard error" ("stderr") filehandle. For datasets consisting of unpaired reads, the summary might look like this:
20000 reads; of these:
20000 (100.00%) were unpaired; of these:
1249 (6.25%) aligned 0 times
18729 (93.64%) aligned exactly 1 time
22 (0.11%) aligned >1 and <=3 times
0 (0.00%) aligned >3 times
93.75% overall alignment rate
For datasets consisting of pairs, the summary might look like this:
10000 reads; of these:
10000 (100.00%) were paired; of these:
650 (6.50%) aligned concordantly 0 times
8822 (88.22%) aligned concordantly exactly 1 time
338 (3.38%) aligned concordantly >1 and <=3 times
190 (1.90%) aligned concordantly >3 times
650 pairs aligned concordantly 0 times; of these:
33 (5.08%) aligned discordantly 1 time
617 pairs aligned 0 times concordantly or discordantly; of these:
1234 mates make up the pairs; of these:
661 (53.57%) aligned 0 times
572 (46.35%) aligned exactly 1 time
1 (0.08%) aligned >1 and <=3 times
0 (0.00%) aligned >3 times
96.69% overall alignment rate
The indentation indicates how subtotals relate to totals.
bowtie2 executable is actually a Perl wrapper script that calls the compiled
bowtie2-align binary. It is recommended that you always run the
bowtie2 wrapper and not run
bowtie2-align directly.
Use 64-bit version if possible
The 64-bit version of Bowtie 2 is faster than the 32-bit version, owing to its use of 64-bit arithmetic. If possible, download the 64-bit binaries for Bowtie 2 and run on a 64-bit computer. If you are building Bowtie 2 from sources, you may need to pass the
-m64 option to
g++ to compile the 64-bit version; you can do this by including
BITS=64 in the arguments to the
make command; e.g.:
make BITS=64 bowtie2. To determine whether your version of bowtie is 64-bit or 32-bit, run
bowtie2 --version.
If your computer has multiple processors/cores, use
-p option causes Bowtie 2 to launch a specified number of parallel search threads. Each thread runs on a different processor/core and all threads find alignments in parallel, increasing alignment throughput by approximately a multiple of the number of threads (though in practice, speedup is somewhat worse than linear).
Some Bowtie 2 options specify a function rather than an individual number or setting. In these cases the user specifies three parameters: (a) a function type
F, (b) a constant term
B, and (c) a coefficient
A. The available function types are constant (
C), linear (
L), square-root (
S), and natural log (
G). The parameters are specified as
F,B,A - that is, the function type, the constant term, and the coefficient are separated by commas with no whitespace. The constant term and coefficient may be negative and/or floating-point numbers.
For example, if the function specification is
L,-0.4,-0.6, then the function defined is:
f(x) = -0.4 + -0.6 * x
If the function specification is
G,1,5.4, then the function defined is:
f(x) = 1.0 + 5.4 * ln(x)
See the documentation for the option in question to learn what the parameter
x is for. For example, in the case if the
--score-min option, the function
f(x) sets the minimum alignment score necessary for an alignment to be considered valid, and
x is the read length.
bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r>} -S [<hit>]
The basename of the index for the reference genome. The basename is the name of any of the index files up to but not including the final
Comma-separated list of files containing mate 1s (filename usually includes
Comma-separated list of files containing mate 2s (filename usually includes
Comma-separated list of files containing unpaired reads to be aligned, e.g.
File to write SAM alignments to. By default, alignments are written to the "standard out" or "stdout" filehandle (i.e. the console).
Reads (specified with
Reads (specified with
Reads (specified with
Reads (specified with
The read sequences are given on command line. I.e.
Skip (i.e. do not align) the first
Align the first
Input qualities are ASCII chars equal to the Phred quality plus 33. This is also called the "Phred+33" encoding, which is used by the very latest Illumina pipelines.
Input qualities are ASCII chars equal to the Phred quality plus 64. This is also called the "Phred+64" encoding.
Convert input qualities from Solexa (which can be negative) to Phred (which can't). This scheme was used in older Illumina GA Pipeline versions (prior to 1.3). Default: off.
Quality values are represented in the read input file as space-separated ASCII integers, e.g.,
--end-to-end mode
Same as:
Same as:
Same as:
Same as:
--local mode
Same as:
Same as:
Same as:
Same as:
Sets the number of mismatches to allowed in a seed alignment during multiseed alignment. Can be set to 0, 1 or 2. Note that 2-mismatch mode is heuristic. Setting this higher makes alignment slower (often much slower) but increases sensitivity. Setting
Sets the length of the seed substrings to align during multiseed alignment. Smaller values make alignment slower but more senstive. Default: the
Sets a function governing the interval between seed substrings to use during multiseed alignment. For instance, if the read has 30 characers, and seed length is 10, and the seed interval is 6, the seeds extracted will be:
Since it's best to use longer intervals for longer reads, this parameter sets the interval as a function of the read length, rather than a single one-size-fits-all number. For instance, specifying
Sets a function governing the maximum number of ambiguous characters (usually
"Pads" dynamic programming problems by
Disallow gaps within
When calculating a mismatch penalty, always consider the quality value at the mismatched position to be the highest possible, regardless of the actual value. I.e. input is treated as though all quality values are high. This is also the default behavior when the input doesn't specify quality values (e.g. in
In this mode, Bowtie 2 requires that the entire read align from one end to the other, without any trimming (or "soft clipping") of characters from either end. The match bonus
In this mode, Bowtie 2 does not require that the entire read align from one end to the other. Rather, some characters may be omitted ("soft clipped") from the ends in order to achieve the greatest possible alignment score. The match bonus
Sets the match bonus. In
Sets the maximum mismatch penalty. A number less than or equal to
Sets penalty for positions where the read, reference, or both, contain an ambiguous character such as
Sets the read gap open (
Sets the reference gap open (
Sets a function governing the minimum alignment score needed for an alignment to be considered "valid" (i.e. good enough to report). This is a function of read length. For instance, specifying
In this mode,
In this mode,
The minimum fragment length for valid paired-end alignments. E.g. if
The maximum fragment length for valid paired-end alignments. E.g. if
The upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand. E.g., if
By default, when
By default,
If the mates "dovetail", that is if one mate alignment extends past the beginning of the other such that the wrong mate begins upstream, consider that to be non-concordant. See also: Mates can overlap, contain or dovetail each other. Default: mates can dovetail in a concordant alignment.
If one mate alignment contains the other, consider that to be non-concordant. See also: Mates can overlap, contain or dovetail each other. Default: a mate can contain the other in a concordant alignment.
If one mate alignment overlaps the other at all, consider that to be non-concordant. See also: Mates can overlap, contain or dovetail each other. Default: mates can overlap in a concordant alignment.
Print the wall-clock time required to load the index files and align the reads. This is printed to the "standard error" ("stderr") filehandle. Default: off.
Print nothing besides alignments and serious errors.
Write a new
Suppress SAM header lines (starting with
Override the offrate of the index with
Use memory-mapped I/O to load the index, rather than normal C file I/O. Memory-mapping the index allows many concurrent
Filter out reads for which the QSEQ filter field is non-zero. Only has an effect when read format is
Print version information and quit.
Print usage information and quit.
Following is a brief description of the SAM format as output by
bowtie2. For more details, see the SAM format specification.
By default,
bowtie2 prints a SAM header with
@SQ and
@PG lines. When one or more
--sam-rg arguments are specified,
bowtie2 will also print an
@RG line that includes all user-specified
--sam-rg tokens separated by tabs.
Each subsequnt line describes an alignment or, if the read failed to align, a read. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are:
Name of read that aligned
Sum of all applicable flags. Flags relevant to Bowtie are:
The read is one of a pair
The alignment is one end of a proper paired-end alignment
The read has no reported alignments
The read is one of a pair and has no reported alignments
The alignment is to the reverse reference strand
The other mate in the paired-end alignment is aligned to the reverse reference strand
The read is mate 1 in a pair
The read is mate 2 in a pair
Thus, an unpaired read that aligns to the reverse reference strand will have flag 16. A paired-end read that aligns and is the first mate in the pair will have flag 83 (= 64 + 16 + 2 + 1).
Name of reference sequence where alignment occurs
1-based offset into the forward reference strand where leftmost character of the alignment occurs
Mapping quality
CIGAR string representation of alignment
Name of reference sequence where mate's alignment occurs. Set to
= if the mate's reference sequence is the same as this alignment's, or
* if there is no mate.
1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate.
Inferred fragment size. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if there is no mate.
Read sequence (reverse-complemented if aligned to the reverse strand)
ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the Phred quality scale and the encoding is ASCII-offset by 33 (ASCII char
!), similarly to a FASTQ file.
Optional fields. Fields are tab-separated.
bowtie2 outputs zero or more of these optional fields for each alignment, depending on the type of the alignment:
Alignment score. Can be negative. Can be greater than 0 in
Alignment score for second-best alignment. Can be negative. Can be greater than 0 in
Alignment score for opposite mate in the paired-end alignment. Only present if the SAM record is for a read that aligned as part of a paired-end alignment.
The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read.
The number of mismatches in the alignment. Only present if SAM record is for an aligned read.
The number of gap opens, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
The number of gap extensions, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
The edit distance; that is, the minimal number of one-nucleotide edits (substitutions, insertions and deletions) needed to transform the read string into the reference string. Only present if SAM record is for an aligned read.
Equals 1 if the read is part of a pair that has at least N concordant alignments, where N is the argument specified to
Equals 1 if the read aligned with at least N unpaired alignments, where N is the argument specified to
String indicating reason why the read was filtered out. See also: Filtering. Only appears for reads that were filtered out.
A string representation of the mismatched reference bases in the alignment. See SAM format specification for details. Only present if SAM record is for an aligned read.
bowtie2-build indexer
bowtie2-build builds a Bowtie index from a set of DNA sequences.
bowtie2-build outputs a set of 6 files with suffixes
.rev.1.bt2, and
.rev.2.bt2. These files together constitute the index: they are all that is needed to align reads to that reference. The original sequence FASTA files are no longer used by Bowtie 2 once the index is built.
Bowtie 2's
.bt2 index format is different from Bowtie 1's
.ebwt format, and they are not compatible with each other.
Use of Karkkainen's blockwise algorithm allows
bowtie2-build to trade off between running time and memory usage.
bowtie2-build has three options governing how it makes this trade:
--bmaxdivn, and
--dcv. By default,
bowtie2-build will automatically search for the settings that yield the best running time without exhausting memory. This behavior can be disabled using the
--noauto option.
The indexer provides options pertaining to the "shape" of the index, e.g.
--offrate governs the fraction of Burrows-Wheeler rows that are "marked" (i.e., the density of the suffix-array sample; see the original FM Index paper for details). All of these options are potentially profitable trade-offs depending on the application. They have been set to defaults that are reasonable for most cases according to our experiments. See Performance tuning for details.
bowtie2-build uses 32-bit pointers internally, it can handle up to a theoretical maximum of 2^32-1 (somewhat more than 4 billion) characters in an index, though, with other constraints, the actual ceiling is somewhat less than that. If your reference exceeds 2^32-1 characters,
bowtie2-build will print an error message and abort. To resolve this, divide your reference sequences into smaller batches and/or chunks and build a separate index for each.
If your computer has more than 3-4 GB of memory and you would like to exploit that fact to make index building faster, use a 64-bit version of the
bowtie2-build binary. The 32-bit version of the binary is restricted to using less than 4 GB of memory. If a 64-bit pre-built binary does not yet exist for your platform on the sourceforge download site, you will need to build one from source.
The Bowtie 2 index is based on the FM Index of Ferragina and Manzini, which in turn is based on the Burrows-Wheeler transform. The algorithm used to build the index is based on the blockwise algorithm of Karkkainen.
bowtie2-build [options]* <reference_in> <bt2_base>
A comma-separated list of FASTA files containing the reference sequences to be aligned to, or, if
The basename of the index files to write. By default,
The reference input files (specified as
The reference sequences are given on the command line. I.e.
Disable the default behavior whereby
Use a packed (2-bits-per-nucleotide) representation for DNA strings. This saves memory but makes indexing 2-3 times slower. Default: off. This is configured automatically by default; use
The maximum number of suffixes allowed in a block. Allowing more suffixes per block makes indexing faster, but increases peak memory usage. Setting this option overrides any previous setting for
The maximum number of suffixes allowed in a block, expressed as a fraction of the length of the reference. Setting this option overrides any previous setting for
Disable use of the difference-cover sample. Suffix sorting becomes quadratic-time in the worst case (where the worst case is an extremely repetitive reference). Default: off.
Do not build the
Build only the
To map alignments back to positions on the reference sequences, it's necessary to annotate ("mark") some or all of the Burrows-Wheeler rows with their corresponding location on the genome.
The ftab is the lookup table used to calculate an initial Burrows-Wheeler range with respect to the first
Index only the first
Print usage information and quit.
Print version information and quit.
bowtie2-inspect index inspector
bowtie2-inspect extracts information from a Bowtie index about what kind of index it is and what reference sequences were used to build it. When run without any options, the tool will output a FASTA file containing the sequences of the original references (with all non-
T characters converted to
Ns). It can also be used to extract just the reference sequence names using the
--names option or a more verbose summary using the
--summary option.
bowtie2-inspect [options]* <bt2_base>
The basename of the index to be inspected. The basename is name of any of the index files but with the
When printing FASTA output, output a newline character every
Print reference sequence names, one per line, and quit.
Print a summary that includes information about index settings, as well as the names and lengths of the input sequences. The summary has this format:
Fields are separated by tabs. Colorspace is always set to 0 for Bowtie 2.
Print verbose output (for debugging).
Print version information and quit.
Print usage information and quit.
Bowtie 2 comes with some example files to get you started. The example files are not scientifically significant; we use the Lambda phage reference genome simply because it's short, and the reads were generated by a computer program, not a sequencer. However, these files will let you start running Bowtie 2 and downstream tools right away.
First follow the manual instructions to obtain Bowtie 2. Set the
BT2_HOME environment variable to point to the new Bowtie 2 directory containing the
bowtie2-build and
bowtie2-inspect binaries. This is important, as the
BT2_HOME variable is used in the commands below to refer to that directory.
To create an index for the Lambda phage reference genome included with Bowtie 2, create a new temporary directory (it doesn't matter where), change into that directory, and run:
$BT2_HOME/bowtie2-build $BT2_HOME/example/reference/lambda_virus.fa lambda_virus
The command should print many lines of output then quit. When the command completes, the current directory will contain four new files that all start with
lambda_virus and end with
.rev.1.bt2, and
.rev.2.bt2. These files constitute the index - you're done!
You can use
bowtie2-build to create an index for a set of FASTA files obtained from any source, including sites such as UCSC, NCBI, and Ensembl. When indexing multiple FASTA files, specify all the files using commas to separate file names. For more details on how to create an index with
bowtie2-build, see the manual section on index building. You may also want to bypass this process by obtaining a pre-built index. See using a pre-built index below for an example.
Stay in the directory created in the previous step, which now contains the
lambda_virus index files. Next, run:
$BT2_HOME/bowtie2 -x lambda_virus -U $BT2_HOME/example/reads/reads_1.fq -S eg1.sam
This runs the Bowtie 2 aligner, which aligns a set of unpaired reads to the Lambda phage reference genome using the index generated in the previous step. The alignment results in SAM format are written to the file
eg1.sam, and a short alignment summary is written to the console. (Actually, the summary is written to the "standard error" or "stderr" filehandle, which is typically printed to the console.)
To see the first few lines of the SAM output, run:
head eg1.sam
You will see something like this:
@HD VN:1.0 SO:unsorted
@SQ SN:gi|9626243|ref|NC_001416.1| LN:48502
@PG ID:bowtie2 PN:bowtie2 VN:2.0.0-beta1
r1 0 gi|9626243|ref|NC_001416.1| 18401 37 122M * 0 0 TGAATGCGAACTCCGGGACGCTCAGTAATGTGACGATAGCTGAAAACTGTACGATAAACNGTACGCTGAGGGCAGAAAAAATCGTCGGGGACATTNTAAAGGCGGCGAGCGCGGCTTTTCCG +"@6<:27(F&5)9)"B:%B+A-%5A?2$HCB0B+0=D<7E/<.03#!.F77@6B==?C"7>;))%;,3-$.A06+<-1/@@?,26">=?*@'0;$:;??G+:#+(A?9+10!8!?()?7C> AS:i:-5 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:59G13G21G26 YT:Z:UU YM:i:0 YF:i:
r2 0 gi|9626243|ref|NC_001416.1| 8886 37 275M * 0 0 NTTNTGATGCGGGCTTGTGGAGTTCAGCCGATCTGACTTATGTCATTACCTATGAAATGTGAGGACGCTATGCCTGTACCAAATCCTACAATGCCGGTGAAAGGTGCCGGGATCACCCTGTGGGTTTATAAGGGGATCGGTGACCCCTACGCGAATCCGCTTTCAGACGTTGACTGGTCGCGTCTGGCAAAAGTTAAAGACCTGACGCCCGGCGAACTGACCGCTGAGNCCTATGACGACAGCTATCTCGATGATGAAGATGCAGACTGGACTGC (#!!'+!$""%+(+)'%)%!+!(&++)''"#"#&#"!'!("%'""("+&%$%*%%#$%#%#!)*'(#")(($&$'&%+&#%*)*#*%*')(%+!%%*"$%"#+)$&&+)&)*+!"*)!*!("&&"*#+"&"'(%)*("'!$*!!%$&&&$!!&&"(*"$&"#&!$%'%"#)$#+%*+)!&*)+(""#!)!%*#"*)*')&")($+*%%)!*)!('(%""+%"$##"#+(('!*(($*'!"*('"+)&%#&$+('**$$&+*&!#%)')'(+(!%+ AS:i:-9 XN:i:0 XM:i:8 XO:i:0 XG:i:0 NM:i:8 MD:Z:0A0C0G0A108C23G9T81T46 YT:Z:UU YM:i:0 YF:i:
r3 16 gi|9626243|ref|NC_001416.1| 11599 37 338M * 0 0 GGGCGCGTTACTGGGATGATCGTGAAAAGGCCCGTCTTGCGCTTGAAGCCGCCCGAAAGAAGGCTGAGCAGCAGACTCAAGAGGAGAAAAATGCGCAGCAGCGGAGCGATACCGAAGCGTCACGGCTGAAATATACCGAAGAGGCGCAGAAGGCTNACGAACGGCTGCAGACGCCGCTGCAGAAATATACCGCCCGTCAGGAAGAACTGANCAAGGCACNGAAAGACGGGAAAATCCTGCAGGCGGATTACAACACGCTGATGGCGGCGGCGAAAAAGGATTATGAAGCGACGCTGTAAAAGCCGAAACAGTCCAGCGTGAAGGTGTCTGCGGGCGAT 7F$%6=$:9B@/F'>=?!D?@0(:A*)7/>9C>6#1<6:C(.CC;#.;>;2'$4D:?&B!>689?(0(G7+0=@37F)GG=>?958.D2E04C<E,*AD%G0.%$+A:'H;?8<72:88?E6((CF)6DF#.)=>B>D-="C'B080E'5BH"77':"@70#4%A5=6.2/1>;9"&-H6)=$/0;5E:<8G!@::1?2DC7C*;@*#.1C0.D>H/20,!"C-#,6@%<+<D(AG-).?�.00'@)/F8?B!&"170,)>:?<A7#1(A@0E#&A.*DC.E")AH"+.,5,2>5"2?:G,F"D0B8D-6$65D<D!A/38860.*4;4B<*31?6 AS:i:-22 XN:i:0 XM:i:8 XO:i:0 XG:i:0 NM:i:8 MD:Z:80C4C16A52T23G30A8T76A41 YT:Z:UU YM:i:0 YF:i:
r4 0 gi|9626243|ref|NC_001416.1| 40075 37 184M * 0 0 GGGCCAATGCGCTTACTGATGCGGAATTACGCCGTAAGGCCGCAGATGAGCTTGTCCATATGACTGCGAGAATTAACNGTGGTGAGGCGATCCCTGAACCAGTAAAACAACTTCCTGTCATGGGCGGTAGACCTCTAAATCGTGCACAGGCTCTGGCGAAGATCGCAGAAATCAAAGCTAAGTT (=8B)GD04*G%&4F,1'A>.C&7=F$,+#6!))43C,5/5+)?-/0>/D3=-,2/+.1?@->;)00!'3!7BH$G)HG+ADC'#-9F)7<7"$?&.>0)@5;4,!0-#C!15CF8&HB+B==H>7,/)C5)5*+(F5A%D,EA<(>G9E0>7&/E?4%;#'92)<5+@7:A.(BG@BG86@.G AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:77C106 YT:Z:UU YM:i:0 YF:i:
r5 0 gi|9626243|ref|NC_001416.1| 48010 37 138M * 0 0 GTCAGGAAAGTGGTAAAACTGCAACTCAATTACTGCAATGCCCTCGTAATTAAGTGAATTTACAATATCGTCCTGTTCGGAGGGAAGAACGCGGGATGTTCATTCTTCATCACTTTTAATTGATGTATATGCTCTCTT 9''%<D)A03E1-*7=),:F/0!6,D9:H,<9D%:0B(%'E,(8EFG$E89B$27G8F*2+4,-!,0D5()&=(FGG:5;3*@/.0F-G#5#3->('FDFEG?)5.!)"AGADB3?6(@H(:B<>6!>;>6>G,."?% AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:138 YT:Z:UU YM:i:0 YF:i:
r6 16 gi|9626243|ref|NC_001416.1| 41607 37 72M2D119M * 0 0 TCGATTTGCAAATACCGGAACATCTCGGTAACTGCATATTCTGCATTAAAAAATCAACGCAAAAAATCGGACGCCTGCAAAGATGAGGAGGGATTGCAGCGTGTTTTTAATGAGGTCATCACGGGATNCCATGTGCGTGACGGNCATCGGGAAACGCCAAAGGAGATTATGTACCGAGGAAGAATGTCGCT 1H#G;H"$E*E#&"*)2%66?=9/9'=;4)4/>@%+5#@#$4A*!<D=="8#1*A9BA=:(1+#C&.#(3#H=9E)AC*5,AC#E'536*2?)H14?>9'B=7(3H/B:+A:8%1-+#(E%&$$&14"76D?>7(&20H5%*&CF8!G5B+A4F$7(:"'?0$?G+$)B-?2<0<F=D!38BH,%=8&5@+ AS:i:-13 XN:i:0 XM:i:2 XO:i:1 XG:i:2 NM:i:4 MD:Z:72^TT55C15A47 YT:Z:UU YM:i:0 YF:i:
r7 16 gi|9626243|ref|NC_001416.1| 4692 37 143M * 0 0 TCAGCCGGACGCGGGCGCTGCAGCCGTACTCGGGGATGACCGGTTACAACGGCATTATCGCCCGTCTGCAACAGGCTGCCAGCGATCCGATGGTGGACAGCATTCTGCTCGATATGGACANGCCCGGCGGGATGGTGGCGGGG -"/@*7A0)>2,AAH@&"%B)*5*23B/,)90.B@%=FE,E063C9?,:26$-0:,.,1849'4.;F>FA;76+5&$<C":$!A*,<B,<)@<'85D%C*:)30@85;?.B$05=@95DCDH<53!8G:F:B7/A.E':434> AS:i:-7 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:98G21C22 YT:Z:UU YM:i:0 YF:i:
The first few lines (beginning with
@) are SAM header lines, and the rest of the lines are SAM alignments, one line per read or mate. See the Bowtie 2 manual section on SAM output and the SAM specification for details about how to interpret the SAM file format.
To align paired-end reads included with Bowtie 2, stay in the same directory and run:
$BT2_HOME/bowtie2 -x lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam
This aligns a set of paired-end reads to the reference genome, with results written to the file
To use local alignment to align some longer reads included with Bowtie 2, stay in the same directory and run:
$BT2_HOME/bowtie2 --local -x lambda_virus -U $BT2_HOME/example/reads/longreads.fq -S eg3.sam
This aligns the long reads to the reference genome using local alignment, with results written to the file
SAMtools is a collection of tools for manipulating and analyzing SAM and BAM alignment files. BCFtools is a collection of tools for calling variants and manipulating VCF and BCF files, and it is typically distributed with SAMtools. Using these tools together allows you to get from alignments in SAM format to variant calls in VCF format. This example assumes that
samtools and
bcftools are installed and that the directories containing these binaries are in your PATH environment variable.
If you haven't already done so above, run the paired-end example. Run it in the same directory where you ran the initial index-building step. Here is the command again:
$BT2_HOME/bowtie2 -x lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam
samtools view to convert the SAM file into a BAM file. BAM is a the binary format corresponding to the SAM text format. Run:
samtools view -bS eg2.sam > eg2.bam
samtools sort to convert the BAM file to a sorted BAM file.
samtools sort eg2.bam eg2.sorted
We now have a sorted BAM file called
eg2.sorted.bam. Sorted BAM is a useful format because the alignments are (a) compressed, which is convenient for long-term storage, and (b) sorted, which is conveneint for variant discovery. To generate variant calls in VCF format, run:
samtools mpileup -uf $BT2_HOME/example/reference/lambda_virus.fa eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf
Then to view the variants, run:
bcftools view eg2.raw.bcf
See the official SAMtools guide to Calling SNPs/INDELs with SAMtools/BCFtools for more details and variations on this process.


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Cancer.Net Associate Editor: Melanoma/Skin Cancers
Massachusetts General Hospital Cancer Center, Boston, MA
Dr. Ryan Sullivan is board certified in Medical Oncology and an Attending Physician in the Division of Hematology/Oncology at Massachusetts General Hospital (MGH). He attended Colby College for undergraduate studies and then matriculated to the University of Connecticut Medical School, graduating in 2001. He first trained in Internal Medicine at Mount Auburn Hospital in Cambridge, MA and then at Beth Israel Deaconess Medical Center (BIDMC) in Hematology/Oncology. He joined the MGH Cancer Center in 2011 and serves as a member of the Center for Melanoma and the Teermer Center for Targeted Therapy. He is an Assistant Professor at Harvard Medical School.
Dr. Sullivan is an active clinical and translational investigator whose main areas of interest are the development of novel therapeutic strategies for malignant melanoma, the translation of promising preclinical findings into early stage clinical trials, and the development of predictive biomarkers for investigational and standard treatment approaches. He is the Principal Investigator on multiple phase I clinical trials and is leading a number of biomarker studies in people with melanoma. He has authored more than 70 research articles, abstracts and reviews in the peer-reviewed literature.
Disclosures: Dr. Sullivan has served as a consultant and/or advisor to Astex Pharmaceuticals, Novartis, Biodesix, and Paometheus.
Last Updated: September 2016


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PortCityDaily.com is your source for free news and information in the Wilmington area.
Lonnie "Sonny" Edward McLaughlin, 74, passed away at home, surrounded by his family on Wednesday, February 10, 2016. He was born in Wilmington, to the late Willie Wilbert and Rosa Lee McLaughlin. Known as "Sonny" to family and friends, he was one of five siblings and a loving husband, father, grandfather and brother.
He is survived by his loving wife of 57 years, Mona "Bonnie" McLaughlin; two daughters, Sheila Mallard, and her husband, Jerry, Melissa Picone, and her husband, John; five grandchildren, Ray Walker (Christian), Christopher Walker (Tiffany), Mary Catherine (Shadie), Joseph Mallard, and Kathryn Mallard (Abraham); and nine great-grandchildren, Caleb, Haileigh, Cali, Amneh, Mohammad, Sonny, Adalee, Hakeem and his little man due to arrive in May; two sisters, Jean Connor and Gwen Griffith; and a large, extended family of cousins, nieces, nephews and in-laws.
He worked in residential construction for most of his life starting his first business, McLaughlin Construction, and then years later opening his second business, Faith Home Builders. He grew up loving the water and had a passion for fishing (and telling fish tales). He was deeply devoted and very proud of his family and loved spending time with all of his "babies."
The family would like to extend their deep appreciation to the staff of the Lower Cape Fear Hospice. The compassion and care that he received truly enriched the quality of his life during his final years with us.
To celebrate his life, the family will receive family and friends from 4 to 6 p.m. Sunday, February 14, at Andrews Mortuary Valley Chapel at 4108 S. College Road. A funeral will be held at 11 a.m. on Monday, February 15, at the same location. Burial will follow in Greenlawn Memorial Park.
Condolences to the family may be expressed at Andrews Mortuary & Crematory. Memorials may be made to Lower Cape Fear Hospice at 725-A Wellington Ave. Wilmington, NC 28401.


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Alternatives to "food" for celebrating Birthday
0 answers | Last updated: Sep 08, 2016
Munkinmom asked...
My brother-in-law has a feeding tube, we want to celebrate his birthday and need suggestions in place of food. He aspirates on any food or drink. Help? Suggestions?
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Daily Craig: Bad luck day
It didn't occur to me it was Friday the 13th UNTIL I LOOKED at the calendar. Funny how that works. I also could tell by the calendar it's BASEBALL AWARD TIME. When you reach week 10 of the NF-, MLB feels lonely and to keep the baseball fires burning they spread out the 200 awards they give each off season. JOSE ALTUVE GOT THE SILVER SLUGGER AWARD one day after his GOLDEN GLOVE, I suppose a PLATINUM THING of some sort is next. Colby* Rasmus got the HOLY AWARD. He is coming back to put his new salary of $15.8 million into the local economy. SURE HE IS. He might spend a couple k* a month on an apartment. Dallas is awating his call for the CY YOUNG and Carlos awaits the ROOK OF THE YEAR. Even the mgr. IS UP FOR MGR. OF THE YEAR. What were we waiting for LAST YEAR AT THIS TIME?
Jose had won the AL BATTING TITLE and was buying new sweat socks. JIM CRANE was threatening his staff with death unless the bottom line improved. Jeff Luhnow was hoping all that time in ST. LOUIS would work out and Chris Carter was hoping to only strike out 300 times."We can't improve in every area" said the guy who drives the train at Minute Maid. As we approach another big weekend, LET'S ASK LES ALEXANDER to dress his team in the uniforms of the last champs. The 1995 outfit. The old logo rocked. Ever wonder why so many fans wear old school t's and replicas to the games? BECAUSE THE current ONES DON'T ROCK. I gotta wonder whey they were ever changed. As for the TEXANS? I am out of ideas for the CINCY GAME. I bet they are too. IT will be the second Sunday in a row you don't have to leave church early. The pastor will think he's starting to get to you. We know better.
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Archived Listing - puncak jalil shoplot, Seri Kembangan, RM 650,000
puncak jalil shoplot, Seri Kembangan, For Sale at RM 650,000
is no longer active which means that the listing has expired or the property has been taken off our search result listings.
Active listings that fulfill similar criteria can be viewed towards the end of this page.
puncak jalil shoplot, bukit jalil, ...
33, puj 3/5 tmn puncak jalil, 43300, Selangor
3,300 sq. ft. / RM 19.97 psf
- Asking Price : RM 650,000
- Asking Price psf: : RM 19.97 per sq. ft.
- Occupancy : Vacant
- Furnishing : Unfurnished
- Date Available : 18/07/2010
- Posted Date : 01/09/2011
This coverage information is indicative only and is not a guarantee of service availability at this property. To get the latest coverage information, visitHERE
Description :
"NEW" CF Ready. 2 Storey intermediate shop; 2 1/2 Storey corner lot. Selling Price from RM510k-RM1.5M. Rental from RM2.2k- RM8k. Hurry!! Limited unit available facing main road. The largest commercial hub at Taman Puncak Jalil. Surrounding more than 4000 units of residential; 80% occupied. Near to Equine Park, Bandar Kinrara, Bukit Jalil & Puchong. Suitable for Banks, Restaurants, Salon, Laundry, Bakery, Boutique, Car Servicing & Many other businesses
Facilities :
- Covered Parking
- Salon
- Business Centre
- Mini Market


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text: Network Magic Pro 5.1 With Network Magic Pro 5.1, you can skip the tech support phone calls, online forums and expensive on-site consultants. Network Magic Pro provides do-it-yourself tools to help you set up, manage and secure your network like an IT professional. With point-and-click ease, you can: 1.Connect your devices together in minutes 2.Share Internet connections, printers and files 3.Protect your network with enhanced WPA security capabilities and status alerts 4.Repair your network and Internet connections to stay online and productive 5.Control access to the Internet and track online activity with remote desktop screenshots Code: http://rapidshare.com/files/221764934/Network_Magic_Pro_5.1.By.VICTORIOUS.rar If you are interested say thanks


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Iam very blonde that my eyeslashes barely seen even with maskara , very thin and very short ...i used to put fake eyelashes that worsen the case...more eyeslashes has fallen appart and didnt grow back ...latise should only grow already existing eyeslashes more but not grow it back i guess ? I love the volume pattern effect on the eyes but anyone can see space between the big sized eyelashes...so my theory is true ? I was looking for eyelashes transplant no pics found , any experience or advices ?
Eyelash Transplant Vs Latisse?
Doctor Answers 2
Latisse to Thicken, Darken, and Lengthen
Latisse can help the eyelashes not only lengthen but thicken as well. This will also help with a darkening effect. Eyelash transplant may be an option, but I do not have any recommendations for that.
Latisse vs eyelash transplant
Latisse works on any active hair follicles, so as long as you have active follicles, even if the hairs are light or short, they will grow thicker, darker, and longer. If you've damaged any follicles irreparably with fake lashes and they don't have the ability to grow hairs anymore, then no, Latisse will not help there. I don't have any experience with eyelash transplants, sorry. But Latisse would be much less expensive and have absolutely no downtime, so in my opinion, it's worth a shot to try it for a few months and see how you respond.
These answers are for educational purposes and should not be relied upon as a substitute for medical advice you may receive from your physician. If you have a medical emergency, please call 911. These answers do not constitute or initiate a patient/doctor relationship.


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Welcome to East Coast Log Cabins
Add living space to your home in an attractive and cost effective way - a log cabin makes an excellent annex, home office or self build. Thermally efficient - easy to keep warm. Cosy traditional interior. Ecologically sound - All the wood used is slow grown and is from sustainable Baltic forests.
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Best Service - We take a personal interest in your project to its completion.
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We offer the following services
Bare log cabin kits delivered to your site. The buildings are completed then disassembled for delivery, each component is numbered and a comprehensive plan is provided to enable easy assembly for a competent DIYer.
If you prefer we will build it for you!
A custom design service if one of our plans doesn't quite suit your requirements - simply supply us with a sketch, or telephone us to discuss your plans and we will have proper plans drawn up and give you a fixed price for your building.
Planning permission - usually planning departments view log buildings as mobile homes and planning is not really an issue, however where planning permission is required we have a lot of experience in this field and can provide assistance if required.
So if you are looking for a warm and cosy home, office or any other outbuilding at outstanding value for money, then you are in the right place!


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Jenson Button and Lewis Hamilton admit that they must capitalise on helpful conditions in the next two races if either of them are to have any chance of winning the world championship.
The long straights at Spa this weekend and Monza in the next race should both suit the McLaren over their closest rivals Red Bull and Ferrari. Yet if Button or Hamilton fail to take advantage, the Woking-based team could struggle going into the season's final five races.
Button said: "This is a little window for us. We know these next two races are important for us. They will suit us for one reason or another – lower downforce or aerodynamic parts on the car – but our pace will be a lot closer and we need to make that count before we get back to high downforce tracks.
"It's crazy how close the first five are. It's not been like that for many years. We've got to finish every race from here on. Consistency is important but there is no point being consistently sixth. You have to be fighting for a podium every race."
Hamilton agreed with his team-mate. "It's very likely that this race and the next race [in Monza] are the two where we're going to be closest to them for the rest of the year, based on the car that we have now."
Hamilton finished second and third in today's practice sessions, which were dominated by Ferrari's Fernando Alonso. Button (sixth and seventh) had a less fluent day. Hamilton, however, is determined to remain upbeat. "Deep down I believe it can happen," he said. "Like I've said, there's a long way to go, and we could win this weekend. I just don't know what to expect. But I'm here to fight for the win, so I will keep pushing to the end."
Red Bull, meanwhile, have come under attack for the way they have treated the championship leader Mark Webber. Alan Jones, a former world champion and the last Australian to take the title, has been upset by the way Red Bull have appeared to favour Sebastian Vettel, who remains the narrow favourite to win the title.
Jones, a blunt-talking Aussie from Melbourne who won the championship with Williams in 1980 – the second Australian to do so after Sir Jack Brabham – has been particularly annoyed by the way Red Bull reacted to the crash involving their two drivers in the Turkish grand prix earlier in the summer. The team appeared to blame Webber for the incident even though Vettel was at fault for steering into the Australian's path.
Jones said today: "If they had spoken to me as they did to Mark after the Turkey race, I would have been pissed off. It was bloody ridiculous because I saw the crash and it was obviously Vettel's fault. He blew it. So for [the team principal] Christian Horner and [the consultant] Helmut Marko to come out with what they did really was a bit rich."
Now Jones, 63, who was known as "AJ" in his racing days, is backing Webber – 34 tomorrow – to beat his team-mate, especially if it comes down to a war of nerves.
He said: "Mark has done it the hard way. He's done the hard yards and he can win it now. He's proven. He's got the speed.
"He hasn't had a reliable car in the past but he has now. And if it comes to a psychological battle I would back Mark every time. He has done a lot for motor sport in Australia. What he has do now is get his side of the garage behind him. With a bit of luck he can win the world championship.
"I went to watch to races at Bahrain and Melbourne. Red Bull obviously have a chassis advantage."
But Webber trailed in 18th place in this afternoon's second practice session, having been seventh in the morning.


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PortCityDaily.com is your source for free news and information in the Wilmington area.
WILMINGTON – It's the time of the year when water-loving Wilmington dogs get to take over the Legion Stadium swimming pool to mark the end of summer.
The annual Pooch Plunge will be taking place from Sept. 6-11. Furry friends will get to cool down in the city-owned pool, which is run by the Wilmington Family YMCA, for a few hours on each of those days. The event has become the traditional way for Wilmington to close down its city pools season, which ends on Labor Day.
Admission is $5 per dog per day (cash only) at the pool, located at 2139 Carolina Beach Rd. in the Legion Stadium Complex. According to organizers, "common-sense dog park rules" apply, which include making sure each pooch is up to date on vaccines and not letting aggressive dogs into the event. Owners will be responsible for making sure their four-legged companions are on their best behavior at all times.
The dogs will be allowed into the water at the following times:
Tuesday, Sept. 6 to Friday, Sept. 9, from 4 to 8 p.m.
Saturday, Sept. 10, from 10 a.m. to 4 p.m.
Sunday, Sept. 11, from 12 p.m. to 4 p.m.


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Frankie's got Bey and Jay tickets for Ol' School Thursday!
You wanna win tickets to see Jay-Z and Beyoncé On The Run? Be caller 10 at 5:25 pm today, Thursday, May 1st. Jay and Bey will be at Citizens Bank Park on July 5th. Tickets go sale on Friday May 2nd at www.ticketmaster.com. See you there!</p


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Aeronautical Development Agency (ADA) hiring B.E, B.Tech holders for Scientist/Engineer role
Company Name: Aeronautical Development Agency (ADA)
Position: Scientist/Engineer
Education: BE/B.Tech
Experience: Freshers
Location: Bangalore
Job Description:
ADA is an Autonomous Organization under the Department of Defence Research & Development, Ministry of Defence (MOD), Government of India, entrusted with the design & development of both the Air Force and Naval versions of Light Combat Aircraft (LCA). LCA Programme is a prestigious National Programme with more than 100 major work centres in India contributing to this Programme and the Programme is in an advanced stage. ADA is also entrusted with the design and development of other advanced projects of Government of India. ADA provides challenging and excellent working environment. ADA has advanced and sophisticated facilities for Aircraft Design activities and provides excellent growth potential.
ADA invites applications for SCIENTISTS / ENGINEERS in various disciplines of Engineering for Design, Development, Testing & Evaluation of various Systems / Sub-systems and Project Management from suitable Scientists and Technologists from Universities, Institutes of Technology, Research Institutions, Public Sector Undertakings and other non-Government bodies in India or abroad or Central (including serving officers of Defence Forces) or State Government Departments possessing the required qualifications and experience only.
Details of the number of posts, Pay & Allowances, educational qualifications and nature of experience required / desired are given below :
Name of the Post : Scientist/Engineer 'G' (To be filled on the basis of direct recruitment / absorption)*
No of Post : 01
Essential : At least First class Bachelor's Degree in Engineering / Technology in Aeronautical / Aerospace Engineering only from a recognized University.
Desirable : Master's degree in Engineering / Technology in the subject concerned.
Experience : Essential : Minimum 15 years experience in one or more of the following areas:(I) Systems Engineering with experience on Aircraft Configuration, Configuration design optimization.(ii) Air intake design methodology, model testing and certification; Development of intake operating envelopes.(iii) Wind tunnel testing – design of F&M models, test planning and data analysis for aerodynamic devices and aero data set preparation.(iv) Aircraft performance estimation.(v) Development of software to calculate performance parameters for ASR complianceDesirable : Experience in Aeronautical industry* ADA employees on contract only can apply in this mode.
Remuneration : Rs.37,400 – 67,000/- & Grade Pay Rs.10,000/- (PB-4)(Minimum Pay in the Pay Band + Grade Play : Rs 53,000 /
Name of the Post : Scientist/Engineer 'D'
No of Post : 03
Essential: At least First class Bachelor's Degree in Engineering / Technology in Computer Science / Computer Science & Engineering / Electronics / Electronics & Communication / Electronics & Electrical Engineering only from a recognized University.
Experience : Essential : Minimum 7 years experience in one or more of the following areas:(I) Computer Graphics concepts & graphics Programming: X Windows, QT, GTK, Open GL, VRML, X3D and graphics based software; (ii) UNIX concepts and programming in UNIX / Linux environment, GNU tools, Scripting, Memory Management, Multithreading, Memory Profiler tools etc. SRS, Development (C, C++, VC++), Version control, Test methodologies, source navigation tools etc; (iii) Atleast two Database applications.
Remuneration : Rs.15,600 – 39,100/- & Grade Pay Rs.7,600/- (PB-3)(Minimum Pay in the Pay Band + Grade Pay : Rs.29,500/-)
Name of the Post :Scientist/Engineer 'F'
No of Post : 02
Essential: At least First class Bachelor's Degree in Engineering / Technology in Electrical / Electrical & Electronics Engineering only from a recognized University
Experience : Essential: Minimum 13 years experience in one or more of the following areas:(I) Design, Development, Testing & Certification of Electrical System & its associated LRUs for a Combat Aircraft.(ii) Principles & Practices used in Design & Routing of Aircraft wiring taking into consideration EMI / EMC & Lightning requirements.(iii) Installation of Avionics & Electrical System LRUs, including Bonding requirements.(iv) Co-ordination with other Design work centers and Govt. Certification and Quality Assurance Agencies during Electrical System Testing in the Ground Rig also on Aircraft; (v) System Performance Monitoring Process during flight evaluation.Desirable : Experience in Global Trend & advances in the area of electrical System Design & Testing; 3D CAD Environment for installation of system LRUs and also Routing of Electrical Looms
Remuneration : Rs.37,400 – 67,000/- & Grade Pay Rs.8,900/- (PB-4)(Minimum Pay in the Pay Band + Grade Play : Rs 49,100
General Instructions 1] Only Indian Nationals need apply. 2 Obtaining First Class in Bachelor's Degree from a recognized University in Engineering/Technology is mandatory. No rounding off of marks is permitted. Candidates should be in possession of First class degree certificate awarded by the university at the time of submitting the application. 3 The applicants should ensure that they satisfy the eligibility criteria as on the closing date of online registration. The prescribed Essential Qualifications and Experience are bare minimum and mere possession of the same does not entitle candidates to be called for interview. 4 Educational qualifications and experience are two separate items of requirements. Candidates should possess the prescribed experience in the relevant fields as mentioned under the column 'Nature and area of experience'. The prescribed experience should have been obtained after acquiring the prescribed educational qualifications. 5 The applications will be screened by the Committee based on the requirements and educational qualifications, experience at suitable level, professional achievement, aptitude for design, development, management skills and personal qualities and strength. The candidates should therefore mention all qualifications / experience /achievements/specialization in relevant field including those over and above the minimum qualifications. The appointment will be subject to the completion of formalities like medical examination prescribed for Group 'A' technical posts of Government of India with field service liability and character verification etc. besides verification of SC/ST/OBC/PH certificates wherever applicable. 6 The period of training/ apprenticeship or part time/casual/per diem employment will not be counted as work experience. Maximum weightage of upto 2 years only will be given for the work experience as Research Fellow / Research Associate / Project Assistant etc. 7 One set of copies of the self-attested certificates in proof of qualifications and experience should be brought at the time of interview along with the application form; however, candidates will be required to produce all original certificates for verification. 8 The upper age limit as on closing date is relaxable upto 5 years for serving Central Civilian Govt. employees. The upper age limit as on closing date is relaxable for physically handicapped persons, ex-serviceman / ex SSC officer as per rules. There is no upper age limit if a vacancy is filled on absorption basis. 9 The selected candidates will be required to resign from the previous organization and will not be permitted to join ADA on lien / deputation basis. 10 Candidates working in Government / Public Sector Undertakings, Autonomous organizations / Armed forces including departmental candidates must apply through proper channel / submit application accompanied with No Objection Certificate (NOC) from the current employer. In addition to registration on website, such candidates must forward a hard copy (downloaded from given website) of the application through proper channel, addressed to 'Chief Administrative Officer', Aeronautical Development Agency, P.B.1718, Vimanapura Post, Bangalore – 560 017. Candidates falling in this category, whose applications have not been received through proper channel within 20 days of the closing date of online registration will not be considered for the interview. 11 Scientists/Engineers employed in ADA on contract / adhoc basis may also apply. However, existing Scientists/Engineers employed in ADA on a regular basis need not apply. 12 If any information furnished online is found to be wrong, the candidates will not be permitted for interview. 13 Selected candidates may be posted anywhere in India if considered necessary. 14 Selected candidates will be governed by the 'National Pension System' applicable to Central Govt. employees appointed on or after 01.01.2004. 15 Candidates applying for more than one post should submit separate online application form. 16 The number of vacancies indicated above may vary and ADA reserves the right not to fill up all or any of the posts, if it so decides. 17 The decision of ADA in all matters relating to eligibility, acceptance or rejection of application shall be final and no enquiry or correspondence will be entertained in this connection from any individual or his/her agency. 18 Canvassing in any form will be treated as disqualification. 19 Genuine queries if any, regarding the advertisement, should be sent by e-mail only on [email protected] 20 Applications/Registrations received after the last date or otherwise incomplete may not be considered. No correspondence in this regard will be entertained. 21 For further information please visit ADA web-site www.ada.gov.in
Important dates to remember :
Last date to apply : 07th April 2014
How to Apply:
Get Daily Job Updates through Email – Click here to Subscribe


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Not any more. Niall FitzGerald, co-chairman and chief executive since 1996, has announced his decision to quit at the age of 58, his main task done - but, it must be said, with insufficient time to embark on another. When appointed he was the youngest leader anyone could remember, so when in 1999 he began his restructuring agenda, Path for Growth, those who might otherwise have resisted knew he meant it and had the time.
So will his successor. FitzGerald is leaving in October, rather than taking the few more years that could be his, to give his heir Patrick Cescau the similar luxury of time. For the record, Cescau is the first Frenchman to run the company.
If FitzGerald is to be judged by Path for Growth, the judgment will be favourable. Analysts have been jittery because sales growth of the leading brands has been slower than planned, but on every other measure he has delivered. The result is a business transformed.
Since 1999 the number of brands has been slashed from 1700 and will be down to the 400 target by the end of the year. Leading brands already account for 95% of turnover, up from 75%, and 12 rather than four of these have sales in excess of e1bn (£678m). In five years 138 factories have been closed, 51,800 jobs eliminated and not a single day lost to industrial action.
Even after this rationalisation the scale of the business is still almost too big to contemplate. More than 150 million times a day in 150 countries someone buys a Unilever product. It is massive in the essential mass market areas of hygiene, personal care and nutrition.
The financial improvement has been every bit as dramatic. Operating margins have soared from 11.2% to 16.7%, working capital has been significantly cut and annual free cashflow has grown by an astonishing e1.1bn to e3.9bn.
Given that free cashflow is a valid measure of a business's worth, this translates into a huge increase in value for shareholders. People like me who at times thought FitzGerald's pruning was too brutal and would fail to deliver, were simply wrong.
Cescau's time will be different, because the future needs of the business are different. FitzGerald's plan was to focus on far fewer brands so there would be enough cash for them to be adequately supported and developed. His successor now has to build on this.
It is easy to mock marketing hype and underestimate its use as a touchstone for the business to know where to invest, what to buy and how much to put behind its products. But in an age when loss of reputation can so quickly destroy a company, the converse is also true - if the company behind the brands is trusted it is a real business asset.
Unilever's mission statement has been changed. It now wants to 'add vitality to life ... to help people feel good, look good and get more out of life'. Vitality is the emblematic word and perhaps that is the real testament to the change FitzGerald has achieved.
The words 'Unilever' and 'vitality' can now be used in the same sentence without provoking widespread hoots of derision. That would not have been the case 10 years ago.
End of an era
SHAREHOLDER meetings are not normally emotional occasions. Dull often, bad-tempered occasionally, but yesterday's meeting of Safeway where the shareholders met to ring down the curtain on the supermarket's days of independence was different because it was genuinely emotional.
It was hard to ignore the fact that though the deal with Wm Morrison is probably the right thing to do, it still marked the end of an adventure that began almost 20 years ago when Jimmy Gulliver, on the rebound from being outsmarted by Guinness in his bid for Distillers, bought the British business of Safeway from its then owner KKR and set about creating a new force in supermarket retailing to challenge Sainsbury and Tesco.
Gulliver was the first leader, then it was his lieutenant Alistair Grant and finally the baton passed to the third member of that original team, the now-retiring chairman David Webster.
Safeway's structural weakness ultimately left it unable to defend itself against the onslaught of Tesco and Wal-Mart but not before this remarkable trio - of whom sadly only Webster is still with us - made a major contribution to British retailing and to business life.
- More about:
- Brand Marketing
- Retail
- Safeway
- Supermarkets
- Unilever


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Zeeshanme.com is a 1 year old domain, situated in United States. The domain is linked to the IP address 188.8.131.52 which is provided by the hosting company Liquid Web.
It was registered by Ahsan Sohail , who can be reached at . The site is owned by N/A. Registration details show that it was registered on 08 Jan 2016 through pdr ltd. d/b/a publicdomainregistry.com and will expire on 08 Jan 2017.
The site is being served through Apache.


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The William Decanter
Recycled Glass Decanter
Our William Glass Collection is made from recycled glass. Each glass is unique; the variations in height, color, and bubbles reflecting the individual artisans that handmade each piece. åÊ
- Handmade.
- Imported.
- Material: Mexican Glass
- Color: Tinted Green
Overall Dimensions
- 6.0"Diam x 9.0"H
- 60 oz
General Care
- Dishwasher safe
See more: Dining + Kitchen


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There were two others with me, in the devil's den below the house. They were in similar cages to mine--except much smaller, and were lain on the ground, covering the numerous bruises, and open gashes spilling their fresh blood onto the floor. I was the last man standing--as she'd caught me the day before the last. Aside from my initiation, there wasn't anything she'd done to break me...at least, not yet.
The others dared not moan or cry out, for she would surely hear. My eyes glazed over the two, one hand on an arm or a leg and the other in their biting mouths. Am I next? I'd asked myself over a dozen times since she brought me here, knowing that it wouldn't be answered until she came down those steps again.
Creeeeaaaaak... Clank, clank, clank, click... "Ohhhh, Jaaaackie~!" asked a feminine voice in an overly zealous, sing-song voice that made my bones jump. I put my hands on the bars of my cage and presses my face between them, trying to force my blurred vision to work through the darkness and focus on the sliver of light coming from the open door above.
Down she made her way to us, descending from purgatory to Hell itself. Nay, not even she could make it to paradise, but somehow managed to sneak herself into a place higher still. We were but her lowly distractions and the undertakers of her wrath--those at the highest held no place for pity.
The chains she dragged across the ground rattled, rattled through all of us as one cried out--finally cracking. "Please! No more mistress!" he said in between pitiful sobs. I spared a glance and looked over at the shaking mass, feeling my heart sink deeper into my chest. We both knew what was coming.
From the darkness in front of me, she appeared with a cool smile on her face and the chains hung over her shoulder. She looked to me as if she was confused of the terror in my eyes and almost hurt when I immediately jumped back as she held a hand out to me. "Do you know what we're going to do today, Jackie...?"
I hesitated, backing up to the bars behind me. "...No, mistress." She unlocked the cage and stepped through. I stood there, my whole body shaking and my heart beating wildly.
"Heh," she huffed, taking the chains off her shoulders and I closed my eyes, preparing for the worst. Clank. Slowly, I opened them to find them lying at my feet. I looked down at them and then up at her, a questioning look on my face. "I'm going to show you what happens when someone breaks a rule," she said calmly, gesturing to the chains for me to pick up.
I bent down, grabbed them, and took a moment to marvel at their heft and strength. With these, I could get out of here! I thought, but it was only a brief thought that was quickly swept away with the touch of her hand on my shoulder.
"I've always wanted a right hand man," she whispered into my ear and I bowed my head, ready to be at her service.
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Britain's Samantha Murray has been crowned world modern pentathlon champion after victory in Warsaw The Clitheroe 24-year-old beat the Chinese pair Qian Chen and Wanxia Liang to take gold in the Polish capital.
Murray's win included a new pentathlon world record as she won the 200m freestyle swim in a time of 2min 03.84sec.
Her victory makes her the fourth British woman to achieve the feat following Wendy Norman, Steph Cook and Mhairi Spence. There was also further success for Murray as her efforts helped Britain to win the silver medal in the team competition along with team-mates Kate French and Freyja Prentice.
Murray won bronze at the world championships in Rome two years ago and silver at the London 2012 Olympics – Britain's 65th of the Games.