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10 <h1>Table of Contents</h1>
14 ><a href="#what-is-bowtie"
18 ><a href="#what-isnt-bowtie"
19 >What isn't Bowtie?</a
22 ><a href="#obtaining-bowtie"
26 ><a href="#building-from-source"
27 >Building from source</a
32 ><a href="#the-bowtie-aligner"
38 ><a href="#the--n-alignment-mode"
44 ><a href="#the--v-alignment-mode"
54 ><a href="#reporting-modes"
58 ><a href="#example-1--a"
64 ><a href="#example-2--k-3"
70 ><a href="#example-3--k-6"
76 ><a href="#example-4-default--k-1"
77 >Example 4: default (<code
82 ><a href="#example-5--a---best"
88 ><a href="#example-6--a---best---strata"
90 >-a --best --strata</code
94 ><a href="#example-7--a--m-3"
100 ><a href="#example-8--a--m-5"
106 ><a href="#example-9--a--m-3---best---strata"
108 >-a -m 3 --best --strata</code
114 ><a href="#paired-end-alignment"
115 >Paired-end Alignment</a
118 ><a href="#colorspace-alignment"
119 >Colorspace Alignment</a
122 ><a href="#colorspace-reads"
126 ><a href="#building-a-colorspace-index"
127 >Building a colorspace index</a
130 ><a href="#decoding-colorspace-alignments"
131 >Decoding colorspace alignments</a
134 ><a href="#paired-end-colorspace-alignment"
135 >Paired-end colorspace alignment</a
140 ><a href="#performance-tuning"
141 >Performance Tuning</a
144 ><a href="#command-line"
148 ><a href="#main-arguments"
160 ><a href="#alignment"
164 ><a href="#reporting"
172 ><a href="#colorspace"
180 ><a href="#performance"
192 ><a href="#default-bowtie-output"
198 ><a href="#sam-bowtie-output"
206 ><a href="#the-bowtie-build-indexer"
212 ><a href="#command-line-1"
216 ><a href="#main-arguments-1"
220 ><a href="#options-1"
228 ><a href="#the-bowtie-inspect-index-inspector"
230 >bowtie-inspect</code
234 ><a href="#command-line-2"
238 ><a href="#main-arguments-2"
242 ><a href="#options-2"
253 ! This manual is written in "markdown" format and thus contains some
254 ! distracting clutter encoding information about how to convert to
255 ! HTML. See 'MANUAL' for a clearer version of this document.
257 <h1 id="what-is-bowtie"
262 ><a href="http://bowtie-bio.sf.net"
264 > is an ultrafast, memory-efficient short read aligner geared toward quickly aligning large sets of short DNA sequences (reads) to large genomes. It aligns 35-base-pair reads to the human genome at a rate of 25 million reads per hour on a typical workstation. Bowtie indexes the genome with a <a href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform"
266 > index to keep its memory footprint small: for the human genome, the index is typically about 2.2 GB (for unpaired alignment) or 2.9 GB (for paired-end or colorspace alignment). Multiple processors can be used simultaneously to achieve greater alignment speed. Bowtie can also output alignments in the standard <a href="http://samtools.sourceforge.net/SAM1.pdf"
268 > format, allowing Bowtie to interoperate with other tools supporting SAM, including the <a href="http://samtools.sourceforge.net/"
270 > consensus, SNP, and indel callers. Bowtie runs on the command line under Windows, Mac OS X, Linux, and Solaris.</p
272 ><a href="http://bowtie-bio.sf.net"
274 > also forms the basis for other tools, including <a href="http://tophat.cbcb.umd.edu/"
276 >: a fast splice junction mapper for RNA-seq reads, <a href="http://cufflinks.cbcb.umd.edu/"
278 >: a tool for transcriptome assembly and isoform quantitiation from RNA-seq reads, <a href="http://bowtie-bio.sf.net/crossbow"
280 >: a cloud-computing software tool for large-scale resequencing data,and <a href="http://bowtie-bio.sf.net/myrna"
282 >: a cloud computing tool for calculating differential gene expression in large RNA-seq datasets.</p
284 >If you use <a href="http://bowtie-bio.sf.net"
286 > for your published research, please cite the <a href="http://genomebiology.com/2009/10/3/R25"
289 ><h1 id="what-isnt-bowtie"
291 >What isn't Bowtie?</a
294 >Bowtie is not a general-purpose alignment tool like <a href="http://mummer.sourceforge.net/"
296 >, <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi"
298 > or <a href="http://www.vmatch.de/"
300 >. Bowtie works best when aligning short reads to large genomes, though it supports arbitrarily small reference sequences (e.g. amplicons) and reads as long as 1024 bases. Bowtie is designed to be extremely fast for sets of short reads where (a) many of the reads have at least one good, valid alignment, (b) many of the reads are relatively high-quality, and (c) the number of alignments reported per read is small (close to 1).</p
302 >Bowtie does not yet report gapped alignments; this is future work.</p
303 ><h1 id="obtaining-bowtie"
308 >You may download either Bowtie sources or binaries for your platform from the <a href="https://sourceforge.net/projects/bowtie-bio/files/bowtie/"
310 > section of the Sourceforge project site. Binaries are currently available for Intel architectures (<code
314 >) running Linux, Windows, and Mac OS X.</p
315 ><h2 id="building-from-source"
317 >Building from source</a
320 >Building Bowtie from source requires a GNU-like environment that includes GCC, GNU Make and other basics. It should be possible to build Bowtie on a vanilla Linux or Mac installation. Bowtie can also be built on Windows using <a href="http://www.cygwin.com/"
322 > or <a href="http://www.mingw.org/"
324 >. We recommend <a href="http://www.tdragon.net/recentgcc/"
325 >TDM's MinGW Build</a
326 >. If using <a href="http://www.mingw.org/"
328 >, you must also have <a href="http://www.mingw.org/wiki/msys"
332 >To build Bowtie, extract the sources, change to the extracted directory, and run GNU <code
334 > (usually with the command <code
336 >, but sometimes with <code
338 >) with no arguments. If building with <a href="http://www.mingw.org/"
342 > from the <a href="http://www.mingw.org/wiki/msys"
346 >To support the <a href="#bowtie-options-p"
350 > (multithreading) option, Bowtie needs the <code
352 > library. To compile Bowtie without <code
354 > (which disables <a href="#bowtie-options-p"
359 >make BOWTIE_PTHREADS=0</code
361 ><h1 id="the-bowtie-aligner"
370 > takes an index and a set of reads as input and outputs a list of alignments. Alignments are selected according to a combination of the <a href="#bowtie-options-v"
374 >/<a href="#bowtie-options-n"
378 >/<a href="#bowtie-options-e"
382 >/<a href="#bowtie-options-l"
386 > options (plus the <a href="#bowtie-options-I"
390 >/<a href="#bowtie-options-X"
394 >/<a href="#bowtie-options-fr"
398 >/<a href="#bowtie-options-fr"
402 >/ <a href="#bowtie-options-fr"
406 > options for paired-end alignment), which define which alignments are legal, and the <a href="#bowtie-options-k"
410 >/<a href="#bowtie-options-a"
414 >/<a href="#bowtie-options-m"
418 >/<a href="#bowtie-options-M"
422 >/<a href="#bowtie-options-best"
426 >/<a href="#bowtie-options-strata"
430 > options which define which and how many legal alignments should be reported.</p
432 >By default, Bowtie enforces an alignment policy similar to <a href="http://maq.sf.net"
434 >'s default quality-aware policy (<a href="#bowtie-options-n"
438 > 2 <a href="#bowtie-options-l"
442 > 28 <a href="#bowtie-options-e"
446 > 70). See <a href="#the--n-alignment-mode"
447 >the -n alignment mode</a
448 > section of the manual for details about this mode. But Bowtie can also enforce a simpler end-to-end k-difference policy (e.g. with <a href="#bowtie-options-v"
452 > 2). See <a href="#the--v-alignment-mode"
453 >the -v alignment mode</a
454 > section of the manual for details about that mode. [The -n alignment mode] and <a href="#the--v-alignment-mode"
455 >the -v alignment mode</a
456 > are mutually exclusive.</p
458 >Bowtie works best when aligning short reads to large genomes (e.g. human or mouse), though it supports arbitrarily small reference sequences and reads as long as 1024 bases. Bowtie is designed to be very fast for sets of short reads where a) many reads have at least one good, valid alignment, b) many reads are relatively high-quality, c) the number of alignments reported per read is small (close to 1). These criteria are generally satisfied in the context of modern short-read analyses such as RNA-seq, ChIP-seq, other types of -seq, and mammalian resequencing. You may observe longer running times in other research contexts.</p
462 > is too slow for your application, try some of the performance-tuning hints described in the [Performance Tuning] section below.</p
464 >Alignments involving one or more ambiguous reference characters (<code
472 >, etc.) are considered invalid by Bowtie. This is true only for ambiguous characters in the reference; alignments involving ambiguous characters in the read are legal, subject to the alignment policy. Ambiguous characters in the read mismatch all other characters. Alignments that "fall off" the reference sequence are not considered valid.</p
474 >The process by which <code
476 > chooses an alignment to report is randomized in order to avoid "mapping bias" - the phenomenon whereby an aligner systematically fails to report a particular class of good alignments, causing spurious "holes" in the comparative assembly. Whenever <code
478 > reports a subset of the valid alignments that exist, it makes an effort to sample them randomly. This randomness flows from a simple seeded pseudo-random number generator and is deterministic in the sense that Bowtie will always produce the same results for the same read when run with the same initial "seed" value (see <a href="#bowtie-options-seed"
484 >In the default mode, <code
486 > can exhibit strand bias. Strand bias occurs when input reference and reads are such that (a) some reads align equally well to sites on the forward and reverse strands of the reference, and (b) the number of such sites on one strand is different from the number on the other strand. When this happens for a given read, <code
488 > effectively chooses one strand or the other with 50% probability, then reports a randomly-selected alignment for that read from among the sites on the selected strand. This tends to overassign alignments to the sites on the strand with fewer sites and underassign to sites on the strand with more sites. The effect is mitigated, though it may not be eliminated, when reads are longer or when paired-end reads are used. Running Bowtie in <a href="#bowtie-options-best"
492 > mode eliminates strand bias by forcing Bowtie to select one strand or the other with a probability that is proportional to the number of best sites on the strand.</p
494 >Gapped alignments are not currently supported, but support is planned for a future release.</p
495 ><h2 id="the--n-alignment-mode"
502 >When the <a href="#bowtie-options-n"
506 > option is specified (which is the default), <code
508 > determines which alignments are valid according to the following policy, which is similar to <a href="http://maq.sf.net"
510 >'s default policy.</p
511 ><ol style="list-style-type: decimal;"
514 >Alignments may have no more than <code
516 > mismatches (where <code
518 > is a number 0-3, set with <a href="#bowtie-options-n"
522 >) in the first <code
526 > is a number 5 or greater, set with <a href="#bowtie-options-l"
530 >) on the high-quality (left) end of the read. The first <code
532 > bases are called the "seed".</p
536 >The sum of the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
540 > mismatched positions (not just in the seed) may not exceed <code
542 > (set with <a href="#bowtie-options-e"
546 >). Where qualities are unavailable (e.g. if the reads are from a FASTA file), the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
552 >The <a href="#bowtie-options-n"
556 > option is mutually exclusive with the <a href="#bowtie-options-v"
562 >If there are many possible alignments satisfying these criteria, Bowtie gives preference to alignments with fewer mismatches and where the sum from criterion 2 is smaller. When the <a href="#bowtie-options-best"
566 > option is specified, Bowtie guarantees the reported alignment(s) are "best" in terms of these criteria (criterion 1 has priority), and that the alignments are reported in best-to-worst order. Bowtie is somewhat slower when <a href="#bowtie-options-best"
572 >Note that <a href="http://maq.sf.net"
574 > internally rounds base qualities to the nearest 10 and rounds qualities greater than 30 to 30. To maintain compatibility, Bowtie does the same. Rounding can be suppressed with the <a href="#bowtie-options-nomaqround"
580 >Bowtie is not fully sensitive in <a href="#bowtie-options-n"
584 > 2 and <a href="#bowtie-options-n"
588 > 3 modes by default. In these modes Bowtie imposes a "backtracking limit" to limit effort spent trying to find valid alignments for low-quality reads unlikely to have any. This may cause bowtie to miss some legal 2- and 3-mismatch alignments. The limit is set to a reasonable default (125 without <a href="#bowtie-options-best"
592 >, 800 with <a href="#bowtie-options-best"
596 >), but the user may decrease or increase the limit using the <a href="#bowtie-options-maxbts"
600 > and/or <a href="#bowtie-options-y"
604 > options. <a href="#bowtie-options-y"
608 > mode is relatively slow but guarantees full sensitivity.</p
609 ><h2 id="the--v-alignment-mode"
616 >In <a href="#bowtie-options-v"
620 > mode, alignments may have no more than <code
622 > mismatches, where <code
624 > may be a number from 0 through 3 set using the <a href="#bowtie-options-v"
628 > option. Quality values are ignored. The <a href="#bowtie-options-v"
632 > option is mutually exclusive with the <a href="#bowtie-options-n"
638 >If there are many legal alignments, Bowtie gives preference to alignments with fewer mismatches. When the <a href="#bowtie-options-best"
642 > option is specified, Bowtie guarantees the reported alignment(s) are "best" in terms of the number of mismatches, and that the alignments are reported in best-to-worst order. Bowtie is somewhat slower when <a href="#bowtie-options-best"
652 >In <a href="#the--n-alignment-mode"
653 >the -n alignment mode</a
654 >, an alignment's "stratum" is defined as the number of mismatches in the "seed" region, i.e. the leftmost <code
658 > is set with the <a href="#bowtie-options-l"
662 > option. In <a href="#the--v-alignment-mode"
663 >the -v alignment mode</a
664 >, an alignment's stratum is defined as the total number of mismatches in the entire alignment. Some of Bowtie's options (e.g. <a href="#bowtie-options-strata"
668 > and <a href="#bowtie-options-m"
672 > use the notion of "stratum" to limit or expand the scope of reportable alignments.</p
673 ><h2 id="reporting-modes"
678 >With the <a href="#bowtie-options-k"
682 >, <a href="#bowtie-options-a"
686 >, <a href="#bowtie-options-m"
690 >, <a href="#bowtie-options-M"
694 >, <a href="#bowtie-options-best"
698 > and <a href="#bowtie-options-strata"
702 > options, the user can flexibily select which alignments are reported. Below we demonstrate a few ways in which these options can be combined. All examples are using the <code
704 > index packaged with Bowtie. The <a href="#bowtie-options-suppress"
708 > option is used to keep the output concise and some output is elided for clarity.</p
709 ><h3 id="example-1--a"
717 >$ ./bowtie -a -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
718 - gi|110640213|ref|NC_008253.1| 148810 10:A>G,13:C>G
719 - gi|110640213|ref|NC_008253.1| 2852852 8:T>A
720 - gi|110640213|ref|NC_008253.1| 4930433 4:G>T,6:C>G
721 - gi|110640213|ref|NC_008253.1| 905664 6:A>G,7:G>T
722 + gi|110640213|ref|NC_008253.1| 1093035 2:T>G,15:A>T
726 >Specifying <a href="#bowtie-options-a"
730 > instructs bowtie to report <em
732 > valid alignments, subject to the alignment policy: <a href="#bowtie-options-v"
736 > 2. In this case, bowtie finds 5 inexact hits in the E. coli genome; 1 hit (the 2nd one listed) has 1 mismatch, and the other 4 hits have 2 mismatches. Four are on the reverse reference strand and one is on the forward strand. Note that they are not listed in best-to-worst order.</p
737 ><h3 id="example-2--k-3"
745 >$ ./bowtie -k 3 -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
746 - gi|110640213|ref|NC_008253.1| 148810 10:A>G,13:C>G
747 - gi|110640213|ref|NC_008253.1| 2852852 8:T>A
748 - gi|110640213|ref|NC_008253.1| 4930433 4:G>T,6:C>G
752 >Specifying <a href="#bowtie-options-k"
756 > 3 instructs bowtie to report up to 3 valid alignments. In this case, a total of 5 valid alignments exist (see <a href="#example-1"
760 > reports 3 out of those 5. <a href="#bowtie-options-k"
764 > can be set to any integer greater than 0.</p
765 ><h3 id="example-3--k-6"
773 >$ ./bowtie -k 6 -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
774 - gi|110640213|ref|NC_008253.1| 148810 10:A>G,13:C>G
775 - gi|110640213|ref|NC_008253.1| 2852852 8:T>A
776 - gi|110640213|ref|NC_008253.1| 4930433 4:G>T,6:C>G
777 - gi|110640213|ref|NC_008253.1| 905664 6:A>G,7:G>T
778 + gi|110640213|ref|NC_008253.1| 1093035 2:T>G,15:A>T
782 >Specifying <a href="#bowtie-options-k"
786 > 6 instructs bowtie to report up to 6 valid alignments. In this case, a total of 5 valid alignments exist, so <code
789 ><h3 id="example-4-default--k-1"
791 >Example 4: default (<code
797 >$ ./bowtie -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
798 - gi|110640213|ref|NC_008253.1| 148810 10:A>G,13:C>G
802 >Leaving the reporting options at their defaults causes <code
804 > to report the first valid alignment it encounters. Because <a href="#bowtie-options-best"
808 > was not specified, we are not guaranteed that bowtie will report the best alignment, and in this case it does not (the 1-mismatch alignment from the previous example would have been better). The default reporting mode is equivalent to <a href="#bowtie-options-k"
813 ><h3 id="example-5--a---best"
821 >$ ./bowtie -a --best -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
822 - gi|110640213|ref|NC_008253.1| 2852852 8:T>A
823 + gi|110640213|ref|NC_008253.1| 1093035 2:T>G,15:A>T
824 - gi|110640213|ref|NC_008253.1| 905664 6:A>G,7:G>T
825 - gi|110640213|ref|NC_008253.1| 148810 10:A>G,13:C>G
826 - gi|110640213|ref|NC_008253.1| 4930433 4:G>T,6:C>G
830 >Specifying <a href="#bowtie-options-best"
834 > results in the same alignments being printed as if just <a href="#bowtie-options-a"
838 > had been specified, but they are guaranteed to be reported in best-to-worst order.</p
839 ><h3 id="example-6--a---best---strata"
842 >-a --best --strata</code
847 >$ ./bowtie -a --best --strata -v 2 --suppress 1,5,6,7 e_coli -c ATGCATCATGCGCCAT
848 - gi|110640213|ref|NC_008253.1| 2852852 8:T>A
852 >Specifying <a href="#bowtie-options-strata"
856 > in addition to <a href="#bowtie-options-a"
860 > and <a href="#bowtie-options-best"
866 > to report only those alignments in the best alignment "stratum". The alignments in the best stratum are those having the least number of mismatches (or mismatches just in the "seed" portion of the alignment in the case of <a href="#bowtie-options-n"
870 > mode). Note that if <a href="#bowtie-options-strata"
874 > is specified, <a href="#bowtie-options-best"
878 > must also be specified.</p
879 ><h3 id="example-7--a--m-3"
887 >$ ./bowtie -a -m 3 -v 2 e_coli -c ATGCATCATGCGCCAT
892 >Specifying <a href="#bowtie-options-m"
896 > 3 instructs bowtie to refrain from reporting any alignments for reads having more than 3 reportable alignments. The <a href="#bowtie-options-m"
900 > option is useful when the user would like to guarantee that reported alignments are "unique", for some definition of unique.</p
902 >Example 1 showed that the read has 5 reportable alignments when <a href="#bowtie-options-a"
906 > and <a href="#bowtie-options-v"
910 > 2 are specified, so the <a href="#bowtie-options-m"
914 > 3 limit causes bowtie to output no alignments.</p
915 ><h3 id="example-8--a--m-5"
923 >$ ./bowtie -a -m 5 -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
924 - gi|110640213|ref|NC_008253.1| 148810 10:A>G,13:C>G
925 - gi|110640213|ref|NC_008253.1| 2852852 8:T>A
926 - gi|110640213|ref|NC_008253.1| 4930433 4:G>T,6:C>G
927 - gi|110640213|ref|NC_008253.1| 905664 6:A>G,7:G>T
928 + gi|110640213|ref|NC_008253.1| 1093035 2:T>G,15:A>T
932 >Specifying <a href="#bowtie-options-m"
936 > 5 instructs bowtie to refrain from reporting any alignments for reads having more than 5 reportable alignments. Since the read has exactly 5 reportable alignments, the <a href="#bowtie-options-m"
940 > 5 limit allows <code
942 > to print them as usual.</p
943 ><h3 id="example-9--a--m-3---best---strata"
946 >-a -m 3 --best --strata</code
951 >$ ./bowtie -a -m 3 --best --strata -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
952 - gi|110640213|ref|NC_008253.1| 2852852 8:T>A
956 >Specifying <a href="#bowtie-options-m"
960 > 3 instructs bowtie to refrain from reporting any alignments for reads having more than 3 reportable alignments. As we saw in Example 6, the read has only 1 reportable alignment when <a href="#bowtie-options-a"
964 >, <a href="#bowtie-options-best"
968 > and <a href="#bowtie-options-strata"
972 > are specified, so the <a href="#bowtie-options-m"
976 > 3 limit allows <code
978 > to print that alignment as usual.</p
980 >Intuitively, the <a href="#bowtie-options-m"
984 > option, when combined with the <a href="#bowtie-options-best"
988 > and <a href="#bowtie-options-strata"
992 > options, guarantees a principled, though weaker form of "uniqueness." A stronger form of uniqueness is enforced when <a href="#bowtie-options-m"
996 > is specified but <a href="#bowtie-options-best"
1000 > and <a href="#bowtie-options-strata"
1005 ><h2 id="paired-end-alignment"
1007 >Paired-end Alignment</a
1012 > can align paired-end reads when properly paired read files are specified using the <a href="#command-line"
1016 > and <a href="#command-line"
1020 > options (for pairs of raw, FASTA, or FASTQ read files), or using the <a href="#command-line"
1024 > option (for Tab-delimited read files). A valid paired-end alignment satisfies these criteria:</p
1025 ><ol style="list-style-type: decimal;"
1027 >Both mates have a valid alignment according to the alignment policy defined by the <a href="#bowtie-options-v"
1031 >/<a href="#bowtie-options-n"
1035 >/<a href="#bowtie-options-e"
1039 >/<a href="#bowtie-options-l"
1045 >The relative orientation and position of the mates satisfy the constraints defined by the <a href="#bowtie-options-I"
1049 >/<a href="#bowtie-options-X"
1053 >/<a href="#bowtie-options-fr"
1057 >/<a href="#bowtie-options-fr"
1061 >/<a href="#bowtie-options-fr"
1068 >Policies governing which paired-end alignments are reported for a given read are specified using the <a href="#bowtie-options-k"
1072 >, <a href="#bowtie-options-a"
1076 > and <a href="#bowtie-options-m"
1080 > options as usual. The <a href="#bowtie-options-strata"
1084 > and <a href="#bowtie-options-best"
1088 > options do not apply in paired-end mode.</p
1090 >A paired-end alignment is reported as a pair of mate alignments, both on a separate line, where the alignment for each mate is formatted the same as an unpaired (singleton) alignment. The alignment for the mate that occurs closest to the beginning of the reference sequence (the "upstream" mate) is always printed before the alignment for the downstream mate. Reads files containing paired-end reads will sometimes name the reads according to whether they are the #1 or #2 mates by appending a <code
1094 > suffix to the read name. If no such suffix is present in Bowtie's input, the suffix will be added when Bowtie prints read names in alignments (except in <a href="#bowtie-options-S"
1098 > "SAM" mode, where mate information is encoded in the <code
1100 > field instead).</p
1102 >Finding a valid paired-end alignment where both mates align to repetitive regions of the reference can be very time-consuming. By default, Bowtie avoids much of this cost by imposing a limit on the number of "tries" it makes to match an alignment for one mate with a nearby alignment for the other. The default limit is 100. This causes <code
1104 > to miss some valid paired-end alignments where both mates lie in repetitive regions, but the user may use the <a href="#bowtie-options-pairtries"
1108 > or <a href="#bowtie-options-y"
1112 > options to increase Bowtie's sensitivity as desired.</p
1114 >Paired-end alignments where one mate's alignment is entirely contained within the other's are considered invalid.</p
1116 >When colospace alignment is enabled via <a href="#bowtie-options-C"
1120 >, the default setting for paired-end orientation is <a href="#bowtie-options-fr"
1124 >. This is because most SOLiD datasets have that orientation. When colorspace alignment is not enabled (default), the default setting for orientation is <a href="#bowtie-options-fr"
1128 >, since most Illumina datasets have this orientation. The default can be overriden in either case.</p
1130 >Because Bowtie uses an in-memory representation of the original reference string when finding paired-end alignments, its memory footprint is larger when aligning paired-end reads. For example, the human index has a memory footprint of about 2.2 GB in single-end mode and 2.9 GB in paired-end mode. Note that paired-end and unpaired alignment incur the same memory footprint in colorspace (e.g. human incurs about 2.9 GB)</p
1131 ><h2 id="colorspace-alignment"
1133 >Colorspace Alignment</a
1136 >As of version 0.12.0, <code
1138 > can align colorspace reads against a colorspace index when <a href="#bowtie-options-C"
1142 > is specified. Colorspace is the characteristic output format of Applied Biosystems' SOLiD system. In a colorspace read, each character is a color rather than a nucleotide, where a color encodes a class of dinucleotides. E.g. the color blue encodes any of the dinucleotides: AA, CC, GG, TT. Colorspace has the advantage of (often) being able to distinguish sequencing errors from SNPs once the read has been aligned. See ABI's <a href="http://tinyurl.com/ygnb2gn"
1143 >Principles of Di-Base Sequencing</a
1144 > document for details.</p
1145 ><h3 id="colorspace-reads"
1147 >Colorspace reads</a
1150 >All input formats (FASTA <a href="#bowtie-options-f"
1154 >, FASTQ <a href="#bowtie-options-q"
1158 >, raw <a href="#bowtie-options-r"
1162 >, tab-delimited <a href="#command-line"
1166 >, command-line <a href="#bowtie-options-c"
1170 >) are compatible with colorspace (<a href="#bowtie-options-C"
1174 >). When <a href="#bowtie-options-C"
1178 > is specified, read sequences are treated as colors. Colors may be encoded either as numbers (<code
1186 >=red) or as characters <code
1198 >Some reads include a primer base as the first character; e.g.:</p
1202 T2213120002010301233221223311331
1204 T2302111203131231130300111123220
1211 > is the primer base. <code
1213 > detects and handles primer bases properly (i.e., the primer base and the adjacent color are both trimmed away prior to alignment) as long as the rest of the read is encoded as numbers.</p
1217 > also handles input in the form of parallel <code
1221 > files. Use <a href="#bowtie-options-f"
1225 > to specify the <code
1227 > files and <a href="#bowtie-options-Q"
1231 > (for unpaired reads) or <a href="#bowtie-options-Q1"
1235 >/<a href="#bowtie-options-Q2"
1239 > (for paired-end reads) to specify the corresponding <code
1241 > files. It is not necessary to first convert to FASTQ, though <code
1243 > also handles FASTQ-formatted colorspace reads (with <a href="#bowtie-options-q"
1248 ><h3 id="building-a-colorspace-index"
1250 >Building a colorspace index</a
1253 >A colorspace index is built in the same way as a normal index except that <a href="#bowtie-build-options-C"
1257 > must be specified when running <code
1259 >. If the user attempts to use <code
1261 > without <a href="#bowtie-options-C"
1265 > to align against an index that was built with <a href="#bowtie-options-C"
1269 > (or vice versa), <code
1271 > prints an error message and quits.</p
1272 ><h3 id="decoding-colorspace-alignments"
1274 >Decoding colorspace alignments</a
1277 >Once a colorspace read is aligned, Bowtie decodes the alignment into nucleotides and reports the decoded nucleotide sequence. A principled decoding scheme is necessary because many different possible decodings are usually possible. Finding the true decoding with 100% certainty requires knowing all variants (e.g. SNPs) in the subject's genome beforehand, which is usually not possible. Instead, <code
1279 > employs the approximate decoding scheme described in the <a href="http://bioinformatics.oxfordjournals.org/cgi/content/abstract/25/14/1754"
1281 >. This scheme attempts to distinguish variants from sequencing errors according to their relative likelihood under a model that considers the quality values of the colors and the (configurable) global likelihood of a SNP.</p
1283 >Quality values are also "decoded" so that each reported quality value is a function of the two color qualities overlapping it. Bowtie again adopts the scheme described in the <a href="http://bioinformatics.oxfordjournals.org/cgi/content/abstract/25/14/1754"
1285 >, i.e., the decoded nucleotide quality is either the sum of the overlapping color qualities (when both overlapping colors correspond to bases that match in the alignment), the quality of the matching color minus the quality of the mismatching color, or 0 (when both overlapping colors correspond to mismatches).</p
1287 >For accurate decoding, <a href="#bowtie-options-snpphred"
1291 >/<a href="#bowtie-options-snpfrac"
1295 > should be set according to the user's best guess of the SNP frequency in the subject. The <a href="#bowtie-options-snpphred"
1299 > parameter sets the SNP penalty directly (on the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1301 > scale), whereas <a href="#bowtie-options-snpfrac"
1305 > allows the user to specify the fraction of sites expected to be SNPs; the fraction is then converted to a <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1307 > internally. For the purpose of decoding, the SNP fraction is defined in terms of SNPs per <em
1309 > base. Thus, if the genome is diploid, heterozygous SNPs have half the weight of homozygous SNPs</p
1311 >Note that in <a href="#bowtie-options-S"
1317 > mode, the decoded nucleotide sequence is printed for alignments, but the original color sequence (with <code
1325 >=red) is printed for unaligned reads without any reported alignments. As always, the <a href="#bowtie-options-un"
1329 >, <a href="#bowtie-options-max"
1333 > and <a href="#bowtie-options-al"
1337 > parameters print reads exactly as they appeared in the input file.</p
1338 ><h3 id="paired-end-colorspace-alignment"
1340 >Paired-end colorspace alignment</a
1343 >Like other platforms, SOLiD supports generation of paired-end reads. When colorspace alignment is enabled, the default paired-end orientation setting is <a href="#bowtie-options-fr"
1347 >. This is because most SOLiD datasets have that orientation.</p
1349 >Note that SOLiD-generated read files can have "orphaned" mates; i.e. mates without a correpsondingly-named mate in the other file. To avoid problems due to orphaned mates, SOLiD paired-end output should first be converted to <code
1351 > files with unpaired mates omitted. This can be accomplished using, for example, [Galaxy]'s conversion tool (click "NGS: QC and manipulation", then "SOLiD-to-FASTQ" in the left-hand sidebar).</p
1352 ><h2 id="performance-tuning"
1354 >Performance Tuning</a
1356 ><ol style="list-style-type: decimal;"
1359 >Use 64-bit bowtie if possible</p
1361 >The 64-bit version of Bowtie is substantially (usually more then 50%) faster than the 32-bit version, owing to its use of 64-bit arithmetic. If possible, download the 64-bit binaries for Bowtie and run on a 64-bit computer. If you are building Bowtie from sources, you may need to pass the <code
1365 > to compile the 64-bit version; you can do this by including <code
1367 > in the arguments to the <code
1369 > command; e.g.: <code
1370 >make BITS=64 bowtie</code
1371 >. To determine whether your version of bowtie is 64-bit or 32-bit, run <code
1372 >bowtie --version</code
1377 >If your computer has multiple processors/cores, use <code
1381 >The <a href="#bowtie-options-p"
1385 > option causes Bowtie 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).</p
1389 >If reporting many alignments per read, try tweaking <code
1390 >bowtie-build --offrate</code
1393 >If you are using the <a href="#bowtie-options-k"
1397 >, <a href="#bowtie-options-a"
1401 > or <a href="#bowtie-options-m"
1405 > options and Bowtie is reporting many alignments per read (an average of more than about 10 per read) and you have some memory to spare, using an index with a denser SA sample can speed things up considerably.</p
1407 >To do this, specify a smaller-than-default <a href="#bowtie-build-options-o"
1413 > value when running <code
1415 >. A denser SA sample yields a larger index, but is also particularly effective at speeding up alignment when many alignments are reported per read. For example, decreasing the index's <a href="#bowtie-build-options-o"
1421 > by 1 could as much as double alignment performance, and decreasing by 2 could quadruple alignment performance, etc.</p
1423 >On the other hand, decreasing <a href="#bowtie-build-options-o"
1429 > increases the size of the Bowtie index, both on disk and in memory when aligning reads. At the default <a href="#bowtie-build-options-o"
1435 > of 5, the SA sample for the human genome occupies about 375 MB of memory when aligning reads. Decreasing the <a href="#bowtie-build-options-o"
1441 > by 1 doubles the memory taken by the SA sample, and decreasing by 2 quadruples the memory taken, etc.</p
1445 >If bowtie "thrashes", try increasing <code
1446 >bowtie --offrate</code
1451 > runs very slow on a relatively low-memory machine (having less than about 4 GB of memory), then try setting <code
1453 > <a href="#bowtie-options-o"
1461 > value than the value used to build the index. For example, <code
1463 >'s default <a href="#bowtie-build-options-o"
1469 > is 5 and all pre-built indexes available from the Bowtie website are built with <a href="#bowtie-build-options-o"
1477 > thrashes when querying such an index, try using <code
1479 > <a href="#bowtie-options-o"
1485 > still thrashes, try <code
1487 > <a href="#bowtie-options-o"
1491 > 7, etc. A higher <a href="#bowtie-options-o"
1499 > to use a sparser sample of the suffix array than is stored in the index; this saves memory but makes alignment reporting slower (which is especially slow when using <a href="#bowtie-options-a"
1503 > or large <a href="#bowtie-options-k"
1507 > or <a href="#bowtie-options-m"
1514 ><h2 id="command-line"
1522 >bowtie [options]* <ebwt> {-1 <m1> -2 <m2> | --12 <r> | <s>} [<hit>]
1525 ><h3 id="main-arguments"
1537 >The basename of the index to be searched. The basename is the name of any of the index files up to but not including the final <code
1543 > looks for the specified index first in the current directory, then in the <code
1545 > subdirectory under the directory where the <code
1547 > executable is located, then looks in the directory specified in the <code
1548 >BOWTIE_INDEXES</code
1549 > environment variable.</p
1558 >Comma-separated list of files containing the #1 mates (filename usually includes <code
1560 >), or, if <a href="#bowtie-options-c"
1564 > is specified, the mate sequences themselves. E.g., this might be <code
1565 >flyA_1.fq,flyB_1.fq</code
1566 >, or, if <a href="#bowtie-options-c"
1570 > is specified, this might be <code
1571 >GGTCATCCT,ACGGGTCGT</code
1572 >. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in <code
1574 >. Reads may be a mix of different lengths. If <code
1576 > is specified, <code
1578 > will read the #1 mates from the "standard in" filehandle.</p
1587 >Comma-separated list of files containing the #2 mates (filename usually includes <code
1589 >), or, if <a href="#bowtie-options-c"
1593 > is specified, the mate sequences themselves. E.g., this might be <code
1594 >flyA_2.fq,flyB_2.fq</code
1595 >, or, if <a href="#bowtie-options-c"
1599 > is specified, this might be <code
1600 >GGTCATCCT,ACGGGTCGT</code
1601 >. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in <code
1603 >. Reads may be a mix of different lengths. If <code
1605 > is specified, <code
1607 > will read the #2 mates from the "standard in" filehandle.</p
1616 >Comma-separated list of files containing a mix of unpaired and paired-end reads in Tab-delimited format. Tab-delimited format is a 1-read-per-line format where unpaired reads consist of a read name, sequence and quality string each separated by tabs. A paired-end read consists of a read name, sequnce of the #1 mate, quality values of the #1 mate, sequence of the #2 mate, and quality values of the #2 mate separated by tabs. Quality values can be expressed using any of the scales supported in FASTQ files. Reads may be a mix of different lengths and paired-end and unpaired reads may be intermingled in the same file. If <code
1618 > is specified, <code
1620 > will read the Tab-delimited reads from the "standard in" filehandle.</p
1629 >A comma-separated list of files containing unpaired reads to be aligned, or, if <a href="#bowtie-options-c"
1633 > is specified, the unpaired read sequences themselves. E.g., this might be <code
1634 >lane1.fq,lane2.fq,lane3.fq,lane4.fq</code
1635 >, or, if <a href="#bowtie-options-c"
1639 > is specified, this might be <code
1640 >GGTCATCCT,ACGGGTCGT</code
1641 >. Reads may be a mix of different lengths. If <code
1643 > is specified, Bowtie gets the reads from the "standard in" filehandle.</p
1652 >File to write alignments to. By default, alignments are written to the "standard out" filehandle (i.e. the console).</p
1663 <tr><td id="bowtie-options-q">
1673 >The query input files (specified either as <code
1679 >) are FASTQ files (usually having extension <code
1683 >). This is the default. See also: <a href="#bowtie-options-solexa-quals"
1685 >--solexa-quals</code
1687 > and <a href="#bowtie-options-integer-quals"
1689 >--integer-quals</code
1692 ></td></tr><tr><td id="bowtie-options-f">
1702 >The query input files (specified either as <code
1708 >) are FASTA files (usually having extension <code
1714 > or similar). All quality values are assumed to be 40 on the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1717 ></td></tr><tr><td id="bowtie-options-r">
1727 >The query input files (specified either as <code
1733 >) are Raw files: one sequence per line, without quality values or names. All quality values are assumed to be 40 on the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1736 ></td></tr><tr><td id="bowtie-options-c">
1746 >The query sequences are given on command line. I.e. <code
1751 ><singles></code
1752 > are comma-separated lists of reads rather than lists of read files.</p
1753 ></td></tr><tr><td id="bowtie-options-C">
1764 >Align in colorspace. Read characters are interpreted as colors. The index specified must be a colorspace index (i.e. built with <code
1766 > <a href="#bowtie-build-options-C"
1772 > will print an error message and quit. See <a href="#colorspace-alignment"
1773 >Colorspace alignment</a
1774 > for more details.</p
1775 ></td></tr><tr><td id="bowtie-options-Q">
1781 >-Q/--quals <files>
1786 >Comma-separated list of files containing quality values for corresponding unpaired CSFASTA reads. Use in combination with <a href="#bowtie-options-C"
1790 > and <a href="#bowtie-options-f"
1794 >. <a href="#bowtie-options-integer-quals"
1796 >--integer-quals</code
1798 > is set automatically when <code
1803 ></td></tr><tr><td id="bowtie-options-Q1">
1813 >Comma-separated list of files containing quality values for corresponding CSFASTA #1 mates. Use in combination with <a href="#bowtie-options-C"
1817 >, <a href="#bowtie-options-f"
1821 >, and <a href="#command-line"
1825 >. <a href="#bowtie-options-integer-quals"
1827 >--integer-quals</code
1829 > is set automatically when <code
1832 ></td></tr><tr><td id="bowtie-options-Q2">
1842 >Comma-separated list of files containing quality values for corresponding CSFASTA #2 mates. Use in combination with <a href="#bowtie-options-C"
1846 >, <a href="#bowtie-options-f"
1850 >, and <a href="#command-line"
1854 >. <a href="#bowtie-options-integer-quals"
1856 >--integer-quals</code
1858 > is set automatically when <code
1861 ></td></tr><tr><td id="bowtie-options-s">
1867 >-s/--skip <int>
1872 >Skip (i.e. do not align) the first <code
1874 > reads or pairs in the input.</p
1875 ></td></tr><tr><td id="bowtie-options-u">
1881 >-u/--qupto <int>
1886 >Only align the first <code
1888 > reads or read pairs from the input (after the <a href="#bowtie-options-s"
1894 > reads or pairs have been skipped). Default: no limit.</p
1895 ></td></tr><tr><td id="bowtie-options-5">
1901 >-5/--trim5 <int>
1908 > bases from high-quality (left) end of each read before alignment (default: 0).</p
1909 ></td></tr><tr><td id="bowtie-options-3">
1915 >-3/--trim3 <int>
1922 > bases from low-quality (right) end of each read before alignment (default: 0).</p
1923 ></td></tr><tr><td id="bowtie-options-phred33-quals">
1933 >Input qualities are ASCII chars equal to the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1935 > plus 33. Default: on.</p
1936 ></td></tr><tr><td id="bowtie-options-phred64-quals">
1946 >Input qualities are ASCII chars equal to the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1948 > plus 64. Default: off.</p
1949 ></td></tr><tr><td id="bowtie-options-solexa-quals">
1959 >Convert input qualities from <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1961 > (which can be negative) to <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1963 > (which can't). This is usually the right option for use with (unconverted) reads emitted by GA Pipeline versions prior to 1.3. Default: off.</p
1964 ></td></tr><tr><td id="bowtie-options-solexa1.3-quals">
1974 >Same as <a href="#bowtie-options-phred64-quals"
1976 >--phred64-quals</code
1978 >. This is usually the right option for use with (unconverted) reads emitted by GA Pipeline version 1.3 or later. Default: off.</p
1979 ></td></tr><tr><td id="bowtie-options-integer-quals">
1989 >Quality values are represented in the read input file as space-separated ASCII integers, e.g., <code
1991 >..., rather than ASCII characters, e.g., <code
1993 >.... Integers are treated as being on the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
1995 > scale unless <a href="#bowtie-options-solexa-quals"
1997 >--solexa-quals</code
1999 > is also specified. Default: off.</p
2007 <tr><td id="bowtie-options-v">
2017 >Report alignments with at most <code
2019 > mismatches. <a href="#bowtie-options-e"
2023 > and <a href="#bowtie-options-l"
2027 > options are ignored and quality values have no effect on what alignments are valid. <a href="#bowtie-options-v"
2031 > is mutually exclusive with <a href="#bowtie-options-n"
2036 ></td></tr><tr><td id="bowtie-options-n">
2042 >-n/--seedmms <int>
2047 >Maximum number of mismatches permitted in the "seed", i.e. the first <code
2049 > base pairs of the read (where <code
2051 > is set with <a href="#bowtie-options-l"
2057 >). This may be 0, 1, 2 or 3 and the default is 2. This option is mutually exclusive with the <a href="#bowtie-options-v"
2062 ></td></tr><tr><td id="bowtie-options-e">
2068 >-e/--maqerr <int>
2073 >Maximum permitted total of quality values at <em
2075 > mismatched read positions throughout the entire alignment, not just in the "seed". The default is 70. Like <a href="http://maq.sf.net"
2079 > rounds quality values to the nearest 10 and saturates at 30; rounding can be disabled with <a href="#bowtie-options-nomaqround"
2084 ></td></tr><tr><td id="bowtie-options-l">
2090 >-l/--seedlen <int>
2095 >The "seed length"; i.e., the number of bases on the high-quality end of the read to which the <a href="#bowtie-options-n"
2099 > ceiling applies. The lowest permitted setting is 5 and the default is 28. <code
2101 > is faster for larger values of <a href="#bowtie-options-l"
2106 ></td></tr><tr><td id="bowtie-options-nomaqround">
2116 ><a href="http://maq.sf.net"
2118 > accepts quality values in the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
2120 > scale, but internally rounds values to the nearest 10, with a maximum of 30. By default, <code
2122 > also rounds this way. <a href="#bowtie-options-nomaqround"
2126 > prevents this rounding in <code
2129 ></td></tr><tr><td id="bowtie-options-I">
2135 >-I/--minins <int>
2140 >The minimum insert size for valid paired-end alignments. E.g. if <code
2142 > is specified and a paired-end alignment consists of two 20-bp alignments in the appropriate orientation with a 20-bp gap between them, that alignment is considered valid (as long as <a href="#bowtie-options-X"
2146 > is also satisfied). A 19-bp gap would not be valid in that case. If trimming options <a href="#bowtie-options-3"
2150 > or <a href="#bowtie-options-5"
2154 > are also used, the <a href="#bowtie-options-I"
2158 > constraint is applied with respect to the untrimmed mates. Default: 0.</p
2159 ></td></tr><tr><td id="bowtie-options-X">
2165 >-X/--maxins <int>
2170 >The maximum insert size for valid paired-end alignments. E.g. if <code
2172 > is specified and a paired-end alignment consists of two 20-bp alignments in the proper orientation with a 60-bp gap between them, that alignment is considered valid (as long as <a href="#bowtie-options-I"
2176 > is also satisfied). A 61-bp gap would not be valid in that case. If trimming options <a href="#bowtie-options-3"
2180 > or <a href="#bowtie-options-5"
2184 > are also used, the <code
2186 > constraint is applied with respect to the untrimmed mates, not the trimmed mates. Default: 250.</p
2187 ></td></tr><tr><td id="bowtie-options-fr">
2200 >The upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand. E.g., if <code
2202 > is specified and there is a candidate paired-end alignment where mate1 appears upstream of the reverse complement of mate2 and the insert length constraints are met, that alignment is valid. Also, if mate2 appears upstream of the reverse complement of mate1 and all other constraints are met, that too is valid. <code
2204 > likewise requires that an upstream mate1 be reverse-complemented and a downstream mate2 be forward-oriented. <code
2206 > requires both an upstream mate1 and a downstream mate2 to be forward-oriented. Default: <code
2208 > when <a href="#bowtie-options-C"
2212 > (colorspace alignment) is not specified, <code
2214 > when <a href="#bowtie-options-C"
2219 ></td></tr><tr><td id="bowtie-options-nofw">
2231 > is specified, <code
2233 > will not attempt to align against the forward reference strand. If <code
2235 > is specified, <code
2237 > will not attempt to align against the reverse-complement reference strand. For paired-end reads using <a href="#bowtie-options-fr"
2241 > or <a href="#bowtie-options-fr"
2249 > apply to the forward and reverse-complement pair orientations. I.e. specifying <code
2251 > and <a href="#bowtie-options-fr"
2255 > will only find reads in the R/F orientation where mate 2 occurs upstream of mate 1 with respect to the forward reference strand.</p
2256 ></td></tr><tr><td id="bowtie-options-maxbts">
2266 >The maximum number of backtracks permitted when aligning a read in <a href="#bowtie-options-n"
2270 > 2 or <a href="#bowtie-options-n"
2274 > 3 mode (default: 125 without <a href="#bowtie-options-best"
2278 >, 800 with <a href="#bowtie-options-best"
2282 >). A "backtrack" is the introduction of a speculative substitution into the alignment. Without this limit, the default parameters will sometimes require that <code
2284 > try 100s or 1,000s of backtracks to align a read, especially if the read has many low-quality bases and/or has no valid alignments, slowing bowtie down significantly. However, this limit may cause some valid alignments to be missed. Higher limits yield greater sensitivity at the expensive of longer running times. See also: <a href="#bowtie-options-y"
2291 ></td></tr><tr><td id="bowtie-options-pairtries">
2296 >--pairtries <int>
2301 >For paired-end alignment, this is the maximum number of attempts <code
2303 > will make to match an alignment for one mate up with an alignment for the opposite mate. Most paired-end alignments require only a few such attempts, but pairs where both mates occur in highly repetitive regions of the reference can require significantly more. Setting this to a higher number allows <code
2305 > to find more paired- end alignments for repetitive pairs at the expense of speed. The default is 100. See also: <a href="#bowtie-options-y"
2312 ></td></tr><tr><td id="bowtie-options-y">
2323 >Try as hard as possible to find valid alignments when they exist, including paired-end alignments. This is equivalent to specifying very high values for the <a href="#bowtie-options-maxbts"
2327 > and <a href="#bowtie-options-pairtries"
2331 > options. This mode is generally much slower than the default settings, but can be useful for certain problems. This mode is slower when (a) the reference is very repetitive, (b) the reads are low quality, or (c) not many reads have valid alignments.</p
2332 ></td></tr><tr><td id="bowtie-options-chunkmbs">
2337 >--chunkmbs <int>
2342 >The number of megabytes of memory a given thread is given to store path descriptors in <a href="#bowtie-options-best"
2346 > mode. Best-first search must keep track of many paths at once to ensure it is always extending the path with the lowest cumulative cost. Bowtie tries to minimize the memory impact of the descriptors, but they can still grow very large in some cases. If you receive an error message saying that chunk memory has been exhausted in <a href="#bowtie-options-best"
2350 > mode, try adjusting this parameter up to dedicate more memory to the descriptors. Default: 64.</p
2356 ><table><tr><td id="bowtie-options-k">
2368 > valid alignments per read or pair (default: 1). Validity of alignments is determined by the alignment policy (combined effects of <a href="#bowtie-options-n"
2372 >, <a href="#bowtie-options-v"
2376 >, <a href="#bowtie-options-l"
2380 >, and <a href="#bowtie-options-e"
2384 >). If more than one valid alignment exists and the <a href="#bowtie-options-best"
2388 > and <a href="#bowtie-options-strata"
2392 > options are specified, then only those alignments belonging to the best alignment "stratum" will be reported. Bowtie is designed to be very fast for small <a href="#bowtie-options-k"
2396 > but bowtie can become significantly slower as <a href="#bowtie-options-k"
2400 > increases. If you would like to use Bowtie for larger values of <a href="#bowtie-options-k"
2404 >, consider building an index with a denser suffix-array sample, i.e. specify a smaller <a href="#bowtie-build-options-o"
2410 > when invoking <code
2412 > for the relevant index (see the <a href="#performance-tuning"
2413 >Performance tuning</a
2414 > section for details).</p
2415 ></td></tr><tr><td id="bowtie-options-a">
2426 >Report all valid alignments per read or pair (default: off). Validity of alignments is determined by the alignment policy (combined effects of <a href="#bowtie-options-n"
2430 >, <a href="#bowtie-options-v"
2434 >, <a href="#bowtie-options-l"
2438 >, and <a href="#bowtie-options-e"
2442 >). If more than one valid alignment exists and the <a href="#bowtie-options-best"
2446 > and <a href="#bowtie-options-strata"
2450 > options are specified, then only those alignments belonging to the best alignment "stratum" will be reported. Bowtie is designed to be very fast for small <a href="#bowtie-options-k"
2454 > but bowtie can become significantly slower if <a href="#bowtie-options-a"
2460 > is specified. If you would like to use Bowtie with <a href="#bowtie-options-a"
2464 >, consider building an index with a denser suffix-array sample, i.e. specify a smaller <a href="#bowtie-build-options-o"
2470 > when invoking <code
2472 > for the relevant index (see the <a href="#performance-tuning"
2473 >Performance tuning</a
2474 > section for details).</p
2475 ></td></tr><tr><td id="bowtie-options-m">
2485 >Suppress all alignments for a particular read or pair if more than <code
2487 > reportable alignments exist for it. Reportable alignments are those that would be reported given the <a href="#bowtie-options-n"
2491 >, <a href="#bowtie-options-v"
2495 >, <a href="#bowtie-options-l"
2499 >, <a href="#bowtie-options-e"
2503 >, <a href="#bowtie-options-k"
2507 >, <a href="#bowtie-options-a"
2511 >, <a href="#bowtie-options-best"
2515 >, and <a href="#bowtie-options-strata"
2519 > options. Default: no limit. Bowtie is designed to be very fast for small <a href="#bowtie-options-m"
2523 > but bowtie can become significantly slower for larger values of <a href="#bowtie-options-m"
2527 >. If you would like to use Bowtie for larger values of <a href="#bowtie-options-k"
2531 >, consider building an index with a denser suffix-array sample, i.e. specify a smaller <a href="#bowtie-build-options-o"
2537 > when invoking <code
2539 > for the relevant index (see the <a href="#performance-tuning"
2540 >Performance tuning</a
2541 > section for details).</p
2542 ></td></tr><tr><td id="bowtie-options-M">
2552 >Behaves like <a href="#bowtie-options-m"
2556 > except that if a read has more than <code
2558 > reportable alignments, one is reported at random. In <a href="#default-bowtie-output"
2559 >default output mode</a
2560 >, the selected alignment's 7th column is set to <code
2562 >+1 to indicate the read has at least <code
2564 >+1 valid alignments. In <a href="#bowtie-options-S"
2570 > mode, the selected alignment is given a <code
2572 > (mapping quality) of 0 and the <code
2574 > field is set to <code
2576 >+1. This option requires <a href="#bowtie-options-best"
2580 >; if specified without <a href="#bowtie-options-best"
2584 >, <a href="#bowtie-options-best"
2588 > is enabled automatically.</p
2589 ></td></tr><tr><td id="bowtie-options-best">
2599 >Make Bowtie guarantee that reported singleton alignments are "best" in terms of stratum (i.e. number of mismatches, or mismatches in the seed in the case of <a href="#bowtie-options-n"
2603 > mode) and in terms of the quality values at the mismatched position(s). Stratum always trumps quality; e.g. a 1-mismatch alignment where the mismatched position has <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
2605 > 40 is preferred over a 2-mismatch alignment where the mismatched positions both have <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
2607 > 10. When <a href="#bowtie-options-best"
2611 > is not specified, Bowtie may report alignments that are sub-optimal in terms of stratum and/or quality (though an effort is made to report the best alignment). <a href="#bowtie-options-best"
2615 > mode also removes all strand bias. Note that <a href="#bowtie-options-best"
2619 > does not affect which alignments are considered "valid" by <code
2621 >, only which valid alignments are reported by <code
2623 >. When <a href="#bowtie-options-best"
2627 > is specified and multiple hits are allowed (via <a href="#bowtie-options-k"
2631 > or <a href="#bowtie-options-a"
2635 >), the alignments for a given read are guaranteed to appear in best-to-worst order in <code
2639 > is somewhat slower when <a href="#bowtie-options-best"
2644 ></td></tr><tr><td id="bowtie-options-strata">
2654 >If many valid alignments exist and are reportable (e.g. are not disallowed via the <a href="#bowtie-options-k"
2658 > option) and they fall into more than one alignment "stratum", report only those alignments that fall into the best stratum. By default, Bowtie reports all reportable alignments regardless of whether they fall into multiple strata. When <a href="#bowtie-options-strata"
2662 > is specified, <a href="#bowtie-options-best"
2666 > must also be specified.</p
2675 <tr><td id="bowtie-options-t">
2686 >Print the amount of wall-clock time taken by each phase.</p
2687 ></td></tr><tr><td id="bowtie-options-B">
2693 >-B/--offbase <int>
2698 >When outputting alignments, number the first base of a reference sequence as <code
2701 ></td></tr><tr><td id="bowtie-options-quiet">
2711 >Print nothing besides alignments.</p
2712 ></td></tr><tr><td id="bowtie-options-refout">
2722 >Write alignments to a set of files named <code
2726 > is the 0-padded index of the reference sequence aligned to. This can be a useful way to break up work for downstream analyses when dealing with, for example, large numbers of reads aligned to the assembled human genome. If <code
2728 > is also specified, it will be ignored.</p
2729 ></td></tr><tr><td id="bowtie-options-refidx">
2739 >When a reference sequence is referred to in a reported alignment, refer to it by 0-based index (its offset into the list of references that were indexed) rather than by name.</p
2740 ></td></tr><tr><td id="bowtie-options-al">
2745 >--al <filename>
2750 >Write all reads for which at least one alignment was reported to a file with name <code
2751 ><filename></code
2752 >. Written reads will appear as they did in the input, without any of the trimming or translation of quality values that may have taken place within <code
2754 >. Paired-end reads will be written to two parallel files with <code
2758 > inserted in the filename, e.g., if <code
2759 ><filename></code
2762 >, the #1 and #2 mates that fail to align will be written to <code
2767 ></td></tr><tr><td id="bowtie-options-un">
2772 >--un <filename>
2777 >Write all reads that could not be aligned to a file with name <code
2778 ><filename></code
2779 >. Written reads will appear as they did in the input, without any of the trimming or translation of quality values that may have taken place within Bowtie. Paired-end reads will be written to two parallel files with <code
2783 > inserted in the filename, e.g., if <code
2784 ><filename></code
2787 >, the #1 and #2 mates that fail to align will be written to <code
2788 >unaligned_1.fq</code
2790 >unaligned_2.fq</code
2791 > respectively. Unless <a href="#bowtie-options-max"
2795 > is also specified, reads with a number of valid alignments exceeding the limit set with the <a href="#bowtie-options-m"
2799 > option are also written to <code
2800 ><filename></code
2802 ></td></tr><tr><td id="bowtie-options-max">
2807 >--max <filename>
2812 >Write all reads with a number of valid alignments exceeding the limit set with the <a href="#bowtie-options-m"
2816 > option to a file with name <code
2817 ><filename></code
2818 >. Written reads will appear as they did in the input, without any of the trimming or translation of quality values that may have taken place within <code
2820 >. Paired-end reads will be written to two parallel files with <code
2824 > inserted in the filename, e.g., if <code
2825 ><filename></code
2828 >, the #1 and #2 mates that exceed the <a href="#bowtie-options-m"
2832 > limit will be written to <code
2836 > respectively. These reads are not written to the file specified with <a href="#bowtie-options-un"
2841 ></td></tr><tr><td id="bowtie-options-suppress">
2846 >--suppress <cols>
2851 >Suppress columns of output in the <a href="#default-bowtie-output"
2852 >default output mode</a
2854 >--suppress 1,5,6</code
2855 > is specified, the read name, read sequence, and read quality fields will be omitted. See <a href="#default-bowtie-output"
2856 >Default Bowtie output</a
2857 > for field descriptions. This option is ignored if the output mode is <a href="#bowtie-options-S"
2865 <tr><td id="bowtie-options-fullref">
2875 >Print the full refernce sequence name, including whitespace, in alignment output. By default <code
2877 > prints everything up to but not including the first whitespace.</p
2884 <tr><td id="bowtie-options-snpphred">
2889 >--snpphred <int>
2894 >When decoding colorspace alignments, use <code
2896 > as the SNP penalty. This should be set to the user's best guess of the true ratio of SNPs per base in the subject genome, converted to the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
2898 > scale. E.g., if the user expects about 1 SNP every 1,000 positions, <code
2900 > should be set to 30 (which is also the default). To specify the fraction directly, use <a href="#bowtie-options-snpfrac"
2906 <tr><td id="bowtie-options-snpfrac">
2911 >--snpfrac <dec>
2916 >When decoding colorspace alignments, use <code
2918 > as the estimated ratio of SNPs per base. For best decoding results, this should be set to the user's best guess of the true ratio. <code
2920 > internally converts the ratio to a <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
2922 >, and behaves as if that quality had been set via the <a href="#bowtie-options-snpphred"
2926 > option. Default: 0.001.</p
2928 <tr><td id="bowtie-options-col-cseq">
2938 >If reads are in colorspace and the <a href="#default-bowtie-output"
2939 >default output mode</a
2942 > causes the reads' color sequence to appear in the read-sequence column (column 5) instead of the decoded nucleotide sequence. See the <a href="#decoding-colorspace-alignments"
2943 >Decoding colorspace alignments</a
2944 > section for details about decoding. This option is ignored in <a href="#bowtie-options-S"
2952 <tr><td id="bowtie-options-col-cqual">
2962 >If reads are in colorspace and the <a href="#default-bowtie-output"
2963 >default output mode</a
2966 > causes the reads' original (color) quality sequence to appear in the quality column (column 6) instead of the decoded qualities. See the <a href="#colorspace-alignment"
2967 >Colorspace alignment</a
2968 > section for details about decoding. This option is ignored in <a href="#bowtie-options-S"
2976 <tr><td id="bowtie-options-col-keepends">
2986 >When decoding colorpsace alignments, <code
2988 > trims off a nucleotide and quality from the left and right edges of the alignment. This is because those nucleotides are supported by only one color, in contrast to the middle nucleotides which are supported by two. Specify <code
2989 >--col-keepends</code
2990 > to keep the extreme-end nucleotides and qualities.</p
2999 <tr><td id="bowtie-options-S">
3010 >Print alignments in <a href="http://samtools.sourceforge.net/SAM1.pdf"
3012 > format. See the <a href="#sam-bowtie-output"
3014 > section of the manual for details. To suppress all SAM headers, use <a href="#bowtie-options-sam-nohead"
3018 > in addition to <code
3020 >. To suppress just the <code
3022 > headers (e.g. if the alignment is against a very large number of reference sequences), use <a href="#bowtie-options-sam-nosq"
3026 > in addition to <code
3030 > does not write BAM files directly, but SAM output can be converted to BAM on the fly by piping <code
3033 >samtools view</code
3034 >. <a href="#bowtie-options-S"
3040 > is not compatible with <a href="#bowtie-options-refout"
3045 ></td></tr><tr><td id="bowtie-options-mapq">
3055 >If an alignment is non-repetitive (according to <a href="#bowtie-options-m"
3059 >, <a href="#bowtie-options-strata"
3063 > and other options) set the <code
3065 > (mapping quality) field to this value. See the <a href="http://samtools.sourceforge.net/SAM1.pdf"
3067 > for details about the <code
3069 > field Default: 255.</p
3070 ></td></tr><tr><td id="bowtie-options-sam-nohead">
3080 >Suppress header lines (starting with <code
3082 >) when output is <a href="#bowtie-options-S"
3088 >. This must be specified <em
3090 > <a href="#bowtie-options-S"
3098 > is ignored unless <a href="#bowtie-options-S"
3104 > is also specified.</p
3105 ></td></tr><tr><td id="bowtie-options-sam-nosq">
3117 > header lines when output is <a href="#bowtie-options-S"
3123 >. This must be specified <em
3125 > <a href="#bowtie-options-S"
3133 > is ignored unless <a href="#bowtie-options-S"
3139 > is also specified.</p
3140 ></td></tr><tr><td id="bowtie-options-sam-RG">
3145 >--sam-RG <text>
3152 > (usually of the form <code
3156 >) as a field on the <code
3158 > header line. Specify <code
3160 > multiple times to set multiple fields. See the <a href="http://samtools.sourceforge.net/SAM1.pdf"
3162 > for details about what fields are legal. Note that, if any <code
3164 > fields are set using this option, the <code
3168 > fields must both be among them to make the <code
3170 > line legal according to the <a href="http://samtools.sourceforge.net/SAM1.pdf"
3174 > is ignored unless <a href="#bowtie-options-S"
3180 > is also specified.</p
3182 <h4 id="performance"
3188 <td id="bowtie-options-o">
3195 >-o/--offrate <int>
3200 >Override the offrate of the index with <code
3204 > is greater than the offrate used to build the index, then some row markings are discarded when the index is read into memory. This reduces the memory footprint of the aligner but requires more time to calculate text offsets. <code
3206 > must be greater than the value used to build the index.</p
3207 ></td></tr><tr><td id="bowtie-options-p">
3213 >-p/--threads <int>
3220 > parallel search threads (default: 1). Threads will run on separate processors/cores and synchronize when parsing reads and outputting alignments. Searching for alignments is highly parallel, and speedup is fairly close to linear. This option is only available if <code
3222 > is linked with the <code
3224 > library (i.e. if <code
3225 >BOWTIE_PTHREADS=0</code
3226 > is not specified at build time).</p
3227 ></td></tr><tr><td id="bowtie-options-mm">
3237 >Use memory-mapped I/O to load the index, rather than normal C file I/O. Memory-mapping the index allows many concurrent <code
3239 > processes on the same computer to share the same memory image of the index (i.e. you pay the memory overhead just once). This facilitates memory-efficient parallelization of <code
3241 > in situations where using <a href="#bowtie-options-p"
3245 > is not possible.</p
3246 ></td></tr><tr><td id="bowtie-options-shmem">
3256 >Use shared memory to load the index, rather than normal C file I/O. Using shared memory allows many concurrent bowtie processes on the same computer to share the same memory image of the index (i.e. you pay the memory overhead just once). This facilitates memory-efficient parallelization of <code
3258 > in situations where using <a href="#bowtie-options-p"
3262 > is not desirable. Unlike <a href="#bowtie-options-mm"
3268 > installs the index into shared memory permanently, or until the user deletes the shared memory chunks manually. See your operating system documentation for details on how to manually list and remove shared memory chunks (on Linux and Mac OS X, these commands are <code
3272 >). You may also need to increase your OS's maximum shared-memory chunk size to accomodate larger indexes; see your OS documentation.</p
3278 ><table><tr><td id="bowtie-options-seed">
3290 > as the seed for pseudo-random number generator.</p
3291 ></td></tr><tr><td id="bowtie-options-verbose">
3301 >Print verbose output (for debugging).</p
3302 ></td></tr><tr><td id="bowtie-options-version">
3312 >Print version information and quit.</p
3313 ></td></tr><tr><td id="bowtie-options-h">
3321 >Print usage information and quit.</p
3323 <h2 id="default-bowtie-output"
3332 > outputs one alignment per line. Each line is a collection of 8 fields separated by tabs; from left to right, the fields are:</p
3333 ><ol style="list-style-type: decimal;"
3336 >Name of read that aligned</p
3340 >Reference strand aligned to, <code
3342 > for forward strand, <code
3348 >Name of reference sequence where alignment occurs, or numeric ID if no name was provided</p
3352 >0-based offset into the forward reference strand where leftmost character of the alignment occurs</p
3356 >Read sequence (reverse-complemented if orientation is <code
3360 >If the read was in colorspace, then the sequence shown in this column is the sequence of <em
3361 >decoded nucleotides</em
3362 >, not the original colors. See the <a href="#colorspace-alignment"
3363 >Colorspace alignment</a
3364 > section for details about decoding. To display colors instead, use the <a href="#bowtie-options-col-cseq"
3372 >ASCII-encoded read qualities (reversed if orientation is <code
3374 >). The encoded quality values are on the Phred scale and the encoding is ASCII-offset by 33 (ASCII char <code
3378 >If the read was in colorspace, then the qualities shown in this column are the <em
3379 >decoded qualities</em
3380 >, not the original qualities. See the <a href="#colorspace-alignment"
3381 >Colorspace alignment</a
3382 > section for details about decoding. To display colors instead, use the <a href="#bowtie-options-col-cqual"
3390 >If <a href="#bowtie-options-M"
3394 > was specified and the prescribed ceiling was exceeded for this read, this column contains the value of the ceiling, indicating that at least that many valid alignments were found in addition to the one reported.</p
3396 >Otherwise, this column contains the number of other instances where the same sequence aligned against the same reference characters as were aligned against in the reported alignment. This is <em
3398 > the number of other places the read aligns with the same number of mismatches. The number in this column is generally not a good proxy for that number (e.g., the number in this column may be '0' while the number of other alignments with the same number of mismatches might be large).</p
3402 >Comma-separated list of mismatch descriptors. If there are no mismatches in the alignment, this field is empty. A single descriptor has the format offset:reference-base>read-base. The offset is expressed as a 0-based offset from the high-quality (5') end of the read.</p
3405 ><h2 id="sam-bowtie-output"
3412 >Following is a brief description of the <a href="http://samtools.sourceforge.net/SAM1.pdf"
3414 > format as output by <code
3416 > when the <a href="#bowtie-options-S"
3422 > option is specified. For more details, see the <a href="http://samtools.sourceforge.net/SAM1.pdf"
3423 >SAM format specification</a
3426 >When <a href="#bowtie-options-S"
3432 > is specified, <code
3434 > prints a SAM header with <code
3440 > lines. When one or more <a href="#bowtie-options-sam-RG"
3444 > arguments are specified, <code
3446 > will also print an <code
3448 > line that includes all user-specified <a href="#bowtie-options-sam-RG"
3452 > tokens separated by tabs.</p
3454 >Each subsequnt line corresponds to a read or an alignment. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are:</p
3455 ><ol style="list-style-type: decimal;"
3458 >Name of read that aligned</p
3462 >Sum of all applicable flags. Flags relevant to Bowtie are:</p
3471 >The read is one of a pair</p
3480 >The alignment is one end of a proper paired-end alignment</p
3489 >The read has no reported alignments</p
3498 >The read is one of a pair and has no reported alignments</p
3507 >The alignment is to the reverse reference strand</p
3516 >The other mate in the paired-end alignment is aligned to the reverse reference strand</p
3525 >The read is the first (#1) mate in a pair</p
3534 >The read is the second (#2) mate in a pair</p
3537 >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).</p
3541 >Name of reference sequence where alignment occurs, or ordinal ID if no name was provided</p
3545 >1-based offset into the forward reference strand where leftmost character of the alignment occurs</p
3553 >CIGAR string representation of alignment</p
3557 >Name of reference sequence where mate's alignment occurs. Set to <code
3559 > if the mate's reference sequence is the same as this alignment's, or <code
3561 > if there is no mate.</p
3565 >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.</p
3569 >Inferred insert size. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if there is no mate.</p
3573 >Read sequence (reverse-complemented if aligned to the reverse strand)</p
3577 >ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the <a href="http://en.wikipedia.org/wiki/FASTQ_format#Variations"
3579 > scale and the encoding is ASCII-offset by 33 (ASCII char <code
3581 >), similarly to a <a href="http://en.wikipedia.org/wiki/FASTQ_format"
3587 >Optional fields. Fields are tab-separated. For descriptions of all possible optional fields, see the SAM format specification. <code
3589 > outputs some of these optional fields for each alignment, depending on the type of the alignment:</p
3598 >Aligned read has an edit distance of <code
3609 >Aligned read has an edit distance of <code
3611 > in colorspace. This field is present in addition to the <code
3613 > field in <a href="#bowtie-options-C"
3619 > mode, but is omitted otherwise.</p
3628 >For aligned reads, <code
3630 > is a string representation of the mismatched reference bases in the alignment. See <a href="http://samtools.sourceforge.net/SAM1.pdf"
3632 > format specification for details. For colorspace alignments, <code
3634 > describes the decoded <em
3636 > alignment, not the colorspace alignment.</p
3645 >Aligned read belongs to stratum <code
3647 >. See <a href="#strata"
3649 > for definition.</p
3658 >For a read with no reported alignments, <code
3660 > is 0 if the read had no alignments. If <a href="#bowtie-options-m"
3664 > was specified and the read's alignments were supressed because the <a href="#bowtie-options-m"
3668 > ceiling was exceeded, <code
3670 > equals the <a href="#bowtie-options-m"
3674 > ceiling + 1, to indicate that there were at least that many valid alignments (but all were suppressed). In <a href="#bowtie-options-M"
3678 > mode, if the alignment was randomly selected because the <a href="#bowtie-options-M"
3682 > ceiling was exceeded, <code
3684 > equals the <a href="#bowtie-options-M"
3688 > ceiling + 1, to indicate that there were at least that many valid alignments (of which one was reported at random).</p
3697 ><h1 id="the-bowtie-build-indexer"
3706 > builds a Bowtie index from a set of DNA sequences. <code
3708 > outputs a set of 6 files with suffixes <code
3720 >. These files together constitute the index: they are all that is needed to align reads to that reference. The original sequence files are no longer used by Bowtie once the index is built.</p
3722 >Use of Karkkainen's [blockwise algorithm] allows <code
3724 > to trade off between running time and memory usage. <code
3726 > has three options governing how it makes this trade: <a href="#bowtie-build-options-p"
3732 >, <a href="#bowtie-build-options-bmax"
3736 >/<a href="#bowtie-build-options-bmaxdivn"
3740 >, and <a href="#bowtie-build-options-dcv"
3744 >. By default, <code
3746 > will automatically search for the settings that yield the best running time without exhausting memory. This behavior can be disabled using the <a href="#bowtie-build-options-a"
3754 >The indexer provides options pertaining to the "shape" of the index, e.g. <a href="#bowtie-build-options-o"
3758 > governs the fraction of <a href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform"
3760 > rows that are "marked" (i.e., the density of the suffix-array sample; see the original <a href="http://portal.acm.org/citation.cfm?id=796543"
3762 > 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.</p
3766 > 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, <code
3768 > 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.</p
3770 >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 <code
3772 > 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.</p
3774 >The Bowtie index is based on the <a href="http://portal.acm.org/citation.cfm?id=796543"
3776 > of Ferragina and Manzini, which in turn is based on the <a href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform"
3778 > transform. The algorithm used to build the index is based on the [blockwise algorithm] of Karkkainen.</p
3779 ><h2 id="command-line-1"
3787 >bowtie-build [options]* <reference_in> <ebwt_base>
3790 ><h3 id="main-arguments-1"
3797 ><reference_in>
3802 >A comma-separated list of FASTA files containing the reference sequences to be aligned to, or, if <a href="#bowtie-build-options-c"
3806 > is specified, the sequences themselves. E.g., <code
3807 ><reference_in></code
3809 >chr1.fa,chr2.fa,chrX.fa,chrY.fa</code
3810 >, or, if <a href="#bowtie-build-options-c"
3814 > is specified, this might be <code
3815 >GGTCATCCT,ACGGGTCGT,CCGTTCTATGCGGCTTA</code
3825 >The basename of the index files to write. By default, <code
3827 > writes files named <code
3836 >NAME.rev.1.ebwt</code
3838 >NAME.rev.2.ebwt</code
3842 ><ebwt_base></code
3857 >The reference input files (specified as <code
3858 ><reference_in></code
3859 >) are FASTA files (usually having extension <code
3866 ></td></tr><tr><td id="bowtie-build-options-c">
3874 >The reference sequences are given on the command line. I.e. <code
3875 ><reference_in></code
3876 > is a comma-separated list of sequences rather than a list of FASTA files.</p
3877 ></td></tr><tr><td id="bowtie-build-options-C">
3885 >Build a colorspace index, to be queried using <code
3887 > <a href="#bowtie-options-C"
3892 ></td></tr><tr><td id="bowtie-build-options-a">
3902 >Disable the default behavior whereby <code
3904 > automatically selects values for the <a href="#bowtie-build-options-bmax"
3908 >, <a href="#bowtie-build-options-dcv"
3912 > and <a href="#bowtie-build-options-p"
3916 > parameters according to available memory. Instead, user may specify values for those parameters. If memory is exhausted during indexing, an error message will be printed; it is up to the user to try new parameters.</p
3917 ></td></tr><tr><td id="bowtie-build-options-p">
3928 >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 <a href="#bowtie-build-options-a"
3934 > to configure manually.</p
3935 ></td></tr><tr><td id="bowtie-build-options-bmax">
3945 >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 <a href="#bowtie-build-options-bmax"
3949 >, or <a href="#bowtie-build-options-bmaxdivn"
3953 >. Default (in terms of the <a href="#bowtie-build-options-bmaxdivn"
3957 > parameter) is <a href="#bowtie-build-options-bmaxdivn"
3961 > 4. This is configured automatically by default; use <a href="#bowtie-build-options-a"
3967 > to configure manually.</p
3968 ></td></tr><tr><td id="bowtie-build-options-bmaxdivn">
3973 >--bmaxdivn <int>
3978 >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 <a href="#bowtie-build-options-bmax"
3982 >, or <a href="#bowtie-build-options-bmaxdivn"
3986 >. Default: <a href="#bowtie-build-options-bmaxdivn"
3990 > 4. This is configured automatically by default; use <a href="#bowtie-build-options-a"
3996 > to configure manually.</p
3997 ></td></tr><tr><td id="bowtie-build-options-dcv">
4009 > as the period for the difference-cover sample. A larger period yields less memory overhead, but may make suffix sorting slower, especially if repeats are present. Must be a power of 2 no greater than 4096. Default: 1024. This is configured automatically by default; use <a href="#bowtie-build-options-a"
4015 > to configure manually.</p
4016 ></td></tr><tr><td id="bowtie-build-options-nodc">
4026 >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.</p
4035 >Do not build the <code
4039 > portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment.</p
4054 > portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment.</p
4055 ></td></tr><tr><td id="bowtie-build-options-o">
4058 >-o/--offrate <int>
4063 >To map alignments back to positions on the reference sequences, it's necessary to annotate ("mark") some or all of the <a href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform"
4065 > rows with their corresponding location on the genome. <a href="#bowtie-build-options-o"
4071 > governs how many rows get marked: the indexer will mark every 2^<code
4073 > rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 5 (every 32nd row is marked; for human genome, annotations occupy about 340 megabytes).</p
4077 >-t/--ftabchars <int>
4082 >The ftab is the lookup table used to calculate an initial <a href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform"
4084 > range with respect to the first <code
4086 > characters of the query. A larger <code
4088 > yields a larger lookup table but faster query times. The ftab has size 4^(<code
4090 >+1) bytes. The default setting is 10 (ftab is 4MB).</p
4091 ></td></tr><tr><td id="bowtie-build-options-ntoa">
4099 >Convert Ns in the reference sequence to As before building the index. By default, Ns are simply excluded from the index and <code
4101 > will not report alignments that overlap them.</p
4102 ></td></tr><tr><td id="bowtie-build-options-big-little">
4110 >Endianness to use when serializing integers to the index file. Default: little-endian (recommended for Intel- and AMD-based architectures).</p
4111 ></td></tr><tr><td id="bowtie-build-options-seed">
4121 > as the seed for pseudo-random number generator.</p
4125 >--cutoff <int>
4130 >Index only the first <code
4132 > bases of the reference sequences (cumulative across sequences) and ignore the rest.</p
4143 > is verbose by default. With this option <code
4145 > will print only error messages.</p
4154 >Print usage information and quit.</p
4163 >Print version information and quit.</p
4165 <h1 id="the-bowtie-inspect-index-inspector"
4168 >bowtie-inspect</code
4169 > index inspector</a
4173 >bowtie-inspect</code
4174 > 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-<code
4182 > characters converted to <code
4184 >s). It can also be used to extract just the reference sequence names using the <a href="#bowtie-build-options-n"
4190 > option or a more verbose summary using the <a href="#bowtie-inspect-options-s"
4197 ><h2 id="command-line-2"
4205 >bowtie-inspect [options]* <ebwt_base>
4208 ><h3 id="main-arguments-2"
4220 >The basename of the index to be inspected. The basename is name of any of the index files but with the <code
4224 > suffix omitted. <code
4225 >bowtie-inspect</code
4226 > first looks in the current directory for the index files, then looks in the <code
4228 > subdirectory under the directory where the currently-running <code
4230 > executable is located, then looks in the directory specified in the <code
4231 >BOWTIE_INDEXES</code
4232 > environment variable.</p
4241 >-a/--across <int>
4246 >When printing FASTA output, output a newline character every <code
4248 > bases (default: 60).</p
4249 ></td></tr><tr><td id="bowtie-build-options-n">
4259 >Print reference sequence names, one per line, and quit.</p
4260 ></td></tr><tr><td id="bowtie-inspect-options-s">
4270 >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:</p
4273 >Colorspace <0 or 1>
4274 SA-Sample 1 in <sample>
4275 FTab-Chars <chars>
4276 Sequence-1 <name> <len>
4277 Sequence-2 <name> <len>
4279 Sequence-N <name> <len>
4283 >Fields are separated by tabs.</p
4284 ></td></tr><tr><td id="bowtie-inspect-options-e">
4294 >By default, when <code
4295 >bowtie-inspect</code
4296 > is run without <a href="#bowtie-options-s"
4300 > or <a href="#bowtie-options-n"
4304 >, it recreates the reference nucleotide sequences using the bit-encoded reference nucleotides kept in the <code
4308 > index files. When <code
4309 >-e/--ebwt-ref</code
4310 > is specified, <code
4311 >bowtie-inspect</code
4312 > recreates the reference sequences from the Burrows-Wheeler-transformed reference sequence in the <code
4314 > file instead. The reference recreation process is much slower when <code
4315 >-e/--ebwt-ref</code
4316 > is specified. Also, when <code
4317 >-e/--ebwt-ref</code
4318 > is specified and the index is in colorspace, the reference is printed in colors (A=blue, C=green, G=orange, T=red).</p
4327 >Print verbose output (for debugging).</p
4336 >Print version information and quit.</p
4345 >Print usage information and quit.</p