What it does
Bowtie is a short read aligner designed to be ultrafast and memory-efficient. It is developed by Ben Langmead and Cole Trapnell. Please cite: Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology 10:R25.
Know what you are doing
There is no such thing (yet) as an automated gearshift in short read mapping. It is all like stick-shift driving in San Francisco. In other words = running this tool with default parameters will probably not give you meaningful results. A way to deal with this is to understand the parameters by carefully reading the documentation and experimenting. Fortunately, Galaxy makes experimenting easy.
Input formats
Bowtie accepts files in Sanger FASTQ format. Use the FASTQ Groomer to prepare your files.
A Note on Built-in Reference Genomes
The default variant for all genomes is "Full", defined as all primary chromosomes (or scaffolds/contigs) including mitochondrial plus associated unmapped, plasmid, and other segments. When only one version of a genome is available in this tool, it represents the default "Full" variant. Some genomes will have more than one variant available. The "Canonical Male" or sometimes simply "Canonical" variant contains the primary chromosomes for a genome. For example a human "Canonical" variant contains chr1-chr22, chrX, chrY, and chrM. The "Canonical Female" variant contains the primary chromosomes excluding chrY.
Outputs
The output is in SAM format, and has the following columns:
Column Description
-------- --------------------------------------------------------
1 QNAME Query (pair) NAME
2 FLAG bitwise FLAG
3 RNAME Reference sequence NAME
4 POS 1-based leftmost POSition/coordinate of clipped sequence
5 MAPQ MAPping Quality (Phred-scaled)
6 CIGAR extended CIGAR string
7 MRNM Mate Reference sequence NaMe ('=' if same as RNAME)
8 MPOS 1-based Mate POSition
9 ISIZE Inferred insert SIZE
10 SEQ query SEQuence on the same strand as the reference
11 QUAL query QUALity (ASCII-33 gives the Phred base quality)
12 OPT variable OPTional fields in the format TAG:VTYPE:VALUE
The flags are as follows:
Flag Description ------ ------------------------------------- 0x0001 the read is paired in sequencing 0x0002 the read is mapped in a proper pair 0x0004 the query sequence itself is unmapped 0x0008 the mate is unmapped 0x0010 strand of the query (1 for reverse) 0x0020 strand of the mate 0x0040 the read is the first read in a pair 0x0080 the read is the second read in a pair 0x0100 the alignment is not primary
It looks like this (scroll sideways to see the entire example):
QNAME FLAG RNAME POS MAPQ CIAGR MRNM MPOS ISIZE SEQ QUAL OPT HWI-EAS91_1_30788AAXX:1:1:1761:343 4 * 0 0 * * 0 0 AAAAAAANNAAAAAAAAAAAAAAAAAAAAAAAAAAACNNANNGAGTNGNNNNNNNGCTTCCCACAGNNCTGG hhhhhhh;;hhhhhhhhhhh^hOhhhhghhhfhhhgh;;h;;hhhh;h;;;;;;;hhhhhhghhhh;;Phhh HWI-EAS91_1_30788AAXX:1:1:1578:331 4 * 0 0 * * 0 0 GTATAGANNAATAAGAAAAAAAAAAATGAAGACTTTCNNANNTCTGNANNNNNNNTCTTTTTTCAGNNGTAG hhhhhhh;;hhhhhhhhhhhhhhhhhhhhhhhhhhhh;;h;;hhhh;h;;;;;;;hhhhhhhhhhh;;hhVh
Bowtie settings
All of the options have a default value. You can change any of them. Most of the options in Bowtie have been implemented here.
Bowtie parameter list
This is an exhaustive list of Bowtie options:
For indexing (bowtie-build):
-a No auto behavior. Disable the default behavior where bowtie automatically
selects values for --bmax/--bmaxdivn/--dcv/--packed parameters according
to the memory available. [off]
--packed Packing. Use a packed representation for DNA strings. [auto]
--bmax INT Suffix maximum. The maximum number of suffixes allowed in a block. [auto]
--bmaxdivn INT Suffix maximum fraction. The maximum number of suffixes allowed in a block
expressed as a fraction of the length of the reference. [4]
--dcv INT Difference-cover sample. Use INT as the period for the difference-cover
sample. [1024]
--nodc INT No difference-cover sample. Disable the difference-cover sample. [off]
-r No reference indexes. Do not build the NAME.3.ebwt and NAME.4.ebwt portions
of the index. Used only for paired-end alignment. [off]
-o Offrate. How many Burrows-Wheeler rows get marked by the indexer. The
indexer will mark every 2^INT rows. The marked rows correspond to rows on
the genome. [5]
-t INT The ftab lookup table used to calculate an initial Burrows-Wheeler range
with respect to the first INT characters of the query. Ftab size is 4^(INT+1)
bytes. [10]
--ntoa N conversion. Convert Ns to As before building the index. Otherwise, Ns are
simply excluded from the index and Bowtie will not find alignments that
overlap them. [off]
--big Endianness. Endianness to use when serializing integers to the index file. [off]
--little Endianness. [--little]
--seed INT Random seed. Use INT as the seed for the pseudo-random number generator. [off]
For aligning (bowtie):
-s INT Skip. Do not align the first INT reads or pairs in the input. [off]
-u INT Align limit. Only align the first INT reads/pairs from the input. [no limit]
-5 INT High-quality trim. Trim INT bases from the high-quality (left) end of each
read before alignment. [0]
-3 INT Low-quality trim. Trim INT bases from the low-quality (right) end of each
read before alignment. [0]
-n INT Mismatch seed. Maximum number of mismatches permitted in the seed (defined
with seed length option). Can be 0, 1, 2, or 3. [2]
-e INT Mismatch quality. Maximum permitted total of quality values at mismatched
read positions. Bowtie rounds quality values to the nearest 10 and saturates
at 30. [70]
-l INT Seed length. The number of bases on the high-quality end of the read to
which the -n ceiling applies. Must be at least 5. [28]
--nomaqround Suppress Maq rounding. Values are internally rounded to the nearest 10 and
saturate at 30. This options turns off that rounding. [off]
-v INT Maq- or SOAP-like alignment policy. This option turns off the default
Maq-like alignment policy in favor of a SOAP-like one. End-to-end alignments
with at most INT mismatches. [off]
-I INT Minimum insert. The minimum insert size for valid paired-end alignments.
Does checking on untrimmed reads if -5 or -3 is used. [0]
-X INT Maximum insert. The maximum insert size for valid paired-end alignments.
Does checking on untrimmed reads if -5 or -3 is used. [250]
--fr Mate orientation. The upstream/downstream mate orientations for a valid
paired-end alignment against the forward reference strand. [--fr]
--rf Mate orientation. [off]
--ff Mate orientation. [off]
--pairtries INT Maximum alignment attempts for paired-end data. [100]
--nofw No forward aligning. Choosing this option means that Bowtie will not attempt
to align against the forward reference strand. [off]
--norc No reverse-complement aligning. Setting this will mean that Bowtie will not
attempt to align against the reverse-complement reference strand. [off]
--un FILENAME Write all reads that could not be aligned to file [off]
--max FILENAME Write all reads with a number of valid alignments exceeding the limit
set with the -m option to file [off]
--maxbts INT Maximum backtracks. The maximum number of backtracks permitted when aligning
a read in -n 2 or -n 3 mode. [125 without --best] [800 with --best]
-y Try hard. Try as hard as possible to find valid alignments when they exist,
including paired-end alignments. [off]
--chunkmbs INT Thread memory. The number of megabytes of memory a given thread is given to
store path descriptors in --best mode. [32]
-k INT Valid alignments. The number of valid alignments per read or pair. [off]
-a All valid alignments. Choosing this means that all valid alignments per read
or pair will be reported. [off]
-m INT Suppress alignments. Suppress all alignments for a particular read or pair
if more than INT reportable alignments exist for it. [no limit]
--best Best mode. Make Bowtie guarantee that reported singleton alignments are
"best" in terms of stratum (the number of mismatches) and quality values at
mismatched position. [off]
--strata Best strata. When running in best mode, report alignments that fall into the
best stratum if there are ones falling into more than one. [off]
-o INT Offrate override. Override the offrate of the index with INT. Some row
markings are discarded when index read into memory. INT must be greater than
the value used to build the index (default: 5). [off]
--seed INT Random seed. Use INT as the seed for the pseudo-random number generator. [off]
--snpphred INT Use INT as the SNP penalty for decoding colorspace alignments. True ratio of
SNPs per base in the subject genome. [see --snpfrac]
--snpfrac DEC Use DEC as the estimated ratio of SNPs per base when decoding colorspace
alignments. [0.001]
--col-keepends Keep the extreme-end nucleotides and qualities when decoding colorspace
alignments. [off]