bwa.1 17.1 KB
 Charles Plessy committed Jan 20, 2011 1 .TH bwa 1 "8 June 2010" "bwa-0.5.8" "Bioinformatics tools"  Charles Plessy committed Jan 20, 2011 2 3 4 5 6 7 8 9 10 11 12 13 14 .SH NAME .PP bwa - Burrows-Wheeler Alignment Tool .SH SYNOPSIS .PP bwa index -a bwtsw database.fasta .PP bwa aln database.fasta short_read.fastq > aln_sa.sai .PP bwa samse database.fasta aln_sa.sai short_read.fastq > aln.sam .PP bwa sampe database.fasta aln_sa1.sai aln_sa2.sai read1.fq read2.fq > aln.sam .PP  Charles Plessy committed Jan 20, 2011 15 bwa bwasw database.fasta long_read.fastq > aln.sam  Charles Plessy committed Jan 20, 2011 16 17 18 19 20 21 22 23 24 25  .SH DESCRIPTION .PP BWA is a fast light-weighted tool that aligns relatively short sequences (queries) to a sequence database (targe), such as the human reference genome. It implements two different algorithms, both based on Burrows-Wheeler Transform (BWT). The first algorithm is designed for short queries up to ~200bp with low error rate (<3%). It does gapped global alignment w.r.t. queries, supports paired-end reads, and is one of the fastest short read alignment algorithms to date while also  Charles Plessy committed Jan 20, 2011 26 visiting suboptimal hits. The second algorithm, BWA-SW, is designed for  Charles Plessy committed Jan 20, 2011 27 28 long reads with more errors. It performs heuristic Smith-Waterman-like alignment to find high-scoring local hits (and thus chimera). On  Charles Plessy committed Jan 20, 2011 29 low-error short queries, BWA-SW is slower and less accurate than the  Charles Plessy committed Jan 20, 2011 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 first algorithm, but on long queries, it is better. .PP For both algorithms, the database file in the FASTA format must be first indexed with the .B index' command, which typically takes a few hours. The first algorithm is implemented via the .B aln' command, which finds the suffix array (SA) coordinates of good hits of each individual read, and the .B samse/sampe' command, which converts SA coordinates to chromosomal coordinate and pairs reads (for sampe'). The second algorithm is invoked by the .B dbtwsw' command. It works for single-end reads only. .SH COMMANDS AND OPTIONS .TP .B index bwa index [-p prefix] [-a algoType] [-c] Index database sequences in the FASTA format. .B OPTIONS: .RS .TP 10 .B -c Build color-space index. The input fast should be in nucleotide space. .TP .B -p STR Prefix of the output database [same as db filename] .TP .B -a STR Algorithm for constructing BWT index. Available options are: .RS .TP .B is IS linear-time algorithm for constructing suffix array. It requires 5.37N memory where N is the size of the database. IS is moderately fast, but does not work with database larger than 2GB. IS is the default algorithm due to its simplicity. The current codes for IS algorithm are reimplemented by Yuta Mori. .TP .B bwtsw Algorithm implemented in BWT-SW. This method works with the whole human genome, but it does not work with database smaller than 10MB and it is usually slower than IS. .RE .RE .TP .B aln bwa aln [-n maxDiff] [-o maxGapO] [-e maxGapE] [-d nDelTail] [-i nIndelEnd] [-k maxSeedDiff] [-l seedLen] [-t nThrds] [-cRN] [-M misMsc] [-O gapOsc] [-E gapEsc] [-q trimQual] > Find the SA coordinates of the input reads. Maximum .I maxSeedDiff differences are allowed in the first .I seedLen subsequence and maximum .I maxDiff differences are allowed in the whole sequence. .B OPTIONS: .RS .TP 10 .B -n NUM Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04] .TP .B -o INT Maximum number of gap opens [1] .TP .B -e INT Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1] .TP .B -d INT Disallow a long deletion within INT bp towards the 3'-end [16] .TP .B -i INT Disallow an indel within INT bp towards the ends [5] .TP .B -l INT Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for -k 2'. [inf] .TP .B -k INT Maximum edit distance in the seed [2] .TP .B -t INT Number of threads (multi-threading mode) [1] .TP .B -M INT Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3] .TP .B -O INT Gap open penalty [11] .TP .B -E INT Gap extension penalty [4] .TP .B -R INT Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp). .TP .B -c Reverse query but not complement it, which is required for alignment in the color space. .TP .B -N Disable iterative search. All hits with no more than .I maxDiff differences will be found. This mode is much slower than the default. .TP .B -q INT Parameter for read trimming. BWA trims a read down to argmax_x{\\sum_{i=x+1}^l(INT-q_i)} if q_l > Generate alignments in the SAM format given single-end reads. Repetitive  Charles Plessy committed Jan 20, 2011 164 hits will be randomly chosen.  Charles Plessy committed Jan 20, 2011 165 166 167 168 169  .B OPTIONS: .RS .TP 10 .B -n INT  Charles Plessy committed Jan 20, 2011 170 171 172 Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Charles Plessy committed Jan 20, 2011 173 174 175 176 .RE .TP .B sampe  Charles Plessy committed Jan 20, 2011 177 178 bwa sampe [-a maxInsSize] [-o maxOcc] [-n maxHitPaired] [-N maxHitDis] [-P] >  Charles Plessy committed Jan 20, 2011 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194  Generate alignments in the SAM format given paired-end reads. Repetitive read pairs will be placed randomly. .B OPTIONS: .RS .TP 8 .B -a INT Maximum insert size for a read pair to be considered being mapped properly. Since 0.4.5, this option is only used when there are not enough good alignment to infer the distribution of insert sizes. [500] .TP .B -o INT Maximum occurrences of a read for pairing. A read with more occurrneces will be treated as a single-end read. Reducing this parameter helps faster pairing. [100000]  Charles Plessy committed Jan 20, 2011 195 196 197 198 199 .TP .B -P Load the entire FM-index into memory to reduce disk operations (base-space reads only). With this option, at least 1.25N bytes of memory are required, where N is the length of the genome.  Charles Plessy committed Jan 20, 2011 200 201 202 203 204 205 206 207 208 209 .TP .B -n INT Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3] .TP .B -N INT Maximum number of alignments to output in the XA tag for disconcordant read pairs (excluding singletons). If a read has more than INT hits, the XA tag will not be written. [10]  Charles Plessy committed Jan 20, 2011 210 211 212 .RE .TP  Charles Plessy committed Jan 20, 2011 213 214 .B bwasw bwa bwasw [-a matchScore] [-b mmPen] [-q gapOpenPen] [-r gapExtPen] [-t  Charles Plessy committed Jan 20, 2011 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 nThreads] [-w bandWidth] [-T thres] [-s hspIntv] [-z zBest] [-N nHspRev] [-c thresCoef] Align query sequences in the file. .B OPTIONS: .RS .TP 10 .B -a INT Score of a match [1] .TP .B -b INT Mismatch penalty [3] .TP .B -q INT Gap open penalty [5] .TP .B -r INT Gap extension penalty. The penalty for a contiguous gap of size k is q+k*r. [2] .TP .B -t INT Number of threads in the multi-threading mode [1] .TP .B -w INT Band width in the banded alignment [33] .TP .B -T INT Minimum score threshold divided by a [37] .TP .B -c FLOAT Coefficient for threshold adjustment according to query length. Given an l-long query, the threshold for a hit to be retained is a*max{T,c*log(l)}. [5.5] .TP .B -z INT Z-best heuristics. Higher -z increases accuracy at the cost of speed. [1] .TP .B -s INT Maximum SA interval size for initiating a seed. Higher -s increases accuracy at the cost of speed. [3] .TP .B -N INT Minimum number of seeds supporting the resultant alignment to skip reverse alignment. [5] .RE .SH SAM ALIGNMENT FORMAT .PP The output of the .B aln' command is binary and designed for BWA use only. BWA outputs the final alignment in the SAM (Sequence Alignment/Map) format. Each line consists of: .TS center box; cb | cb | cb n | l | l . Col Field 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 CIAGR extended CIGAR string 7 MRNM Mate Reference sequence NaMe (=' if same as RNAME) 8 MPOS 1-based Mate POSistion 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 .TE .PP Each bit in the FLAG field is defined as: .TS center box; cb | cb | cb c | l | l . Chr Flag Description _ p 0x0001 the read is paired in sequencing P 0x0002 the read is mapped in a proper pair u 0x0004 the query sequence itself is unmapped U 0x0008 the mate is unmapped r 0x0010 strand of the query (1 for reverse) R 0x0020 strand of the mate 1 0x0040 the read is the first read in a pair 2 0x0080 the read is the second read in a pair s 0x0100 the alignment is not primary f 0x0200 QC failure d 0x0400 optical or PCR duplicate .TE .PP The Please check for the format specification and the tools for post-processing the alignment. BWA generates the following optional fields. Tags starting with X' are specific to BWA. .TS center box; cb | cb cB | l . Tag Meaning _ NM Edit distance MD Mismatching positions/bases AS Alignment score _ X0 Number of best hits X1 Number of suboptimal hits found by BWA XN Number of ambiguous bases in the referenece XM Number of mismatches in the alignment XO Number of gap opens XG Number of gap extentions XT Type: Unique/Repeat/N/Mate-sw  Charles Plessy committed Jan 20, 2011 336 XA Alternative hits; format: (chr,pos,CIGAR,NM;)*  Charles Plessy committed Jan 20, 2011 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 _ XS Suboptimal alignment score XF Support from forward/reverse alignment XE Number of supporting seeds .TE .PP Note that XO and XG are generated by BWT search while the CIGAR string by Smith-Waterman alignment. These two tags may be inconsistent with the CIGAR string. This is not a bug. .SH NOTES ON SHORT-READ ALIGNMENT .SS Alignment Accuracy .PP When seeding is disabled, BWA guarantees to find an alignment containing maximum .I maxDiff differences including .I maxGapO gap opens which do not occur within .I nIndelEnd bp towards either end of the query. Longer gaps may be found if .I maxGapE is positive, but it is not guaranteed to find all hits. When seeding is enabled, BWA further requires that the first .I seedLen subsequence contains no more than .I maxSeedDiff differences. .PP When gapped alignment is disabled, BWA is expected to generate the same alignment as Eland, the Illumina alignment program. However, as BWA change N' in the database sequence to random nucleotides, hits to these random sequences will also be counted. As a consequence, BWA may mark a unique hit as a repeat, if the random sequences happen to be identical to the sequences which should be unqiue in the database. This random behaviour will be avoided in future releases. .PP By default, if the best hit is no so repetitive (controlled by -R), BWA also finds all hits contains one more mismatch; otherwise, BWA finds all equally best hits only. Base quality is NOT considered in evaluating  Charles Plessy committed Jan 20, 2011 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 hits. In paired-end alignment, BWA pairs all hits it found. It further performs Smith-Waterman alignment for unmapped reads with mates mapped to rescue mapped mates, and for high-quality anomalous pairs to fix potential alignment errors. .SS Estimating Insert Size Distribution .PP BWA estimates the insert size distribution per 256*1024 read pairs. It first collects pairs of reads with both ends mapped with a single-end quality 20 or higher and then calculates median (Q2), lower and higher quartile (Q1 and Q3). It estimates the mean and the variance of the insert size distribution from pairs whose insert sizes are within interval [Q1-2(Q3-Q1), Q3+2(Q3-Q1)]. The maximum distance x for a pair considered to be properly paired (SAM flag 0x2) is calculated by solving equation Phi((x-mu)/sigma)=x/L*p0, where mu is the mean, sigma is the standard error of the insert size distribution, L is the length of the genome, p0 is prior of anomalous pair and Phi() is the standard cumulative distribution function. For mapping Illumina short-insert reads to the human genome, x is about 6-7 sigma away from the mean. Quartiles, mean, variance and x will be printed to the standard error output.  Charles Plessy committed Jan 20, 2011 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441  .SS Memory Requirement .PP With bwtsw algorithm, 2.5GB memory is required for indexing the complete human genome sequences. For short reads, the .B aln' command uses ~2.3GB memory and the .B sampe' command uses ~3.5GB. .SS Speed .PP Indexing the human genome sequences takes 3 hours with bwtsw algorithm. Indexing smaller genomes with IS or divsufsort algorithms is several times faster, but requires more memory. .PP Speed of alignment is largely determined by the error rate of the query sequences (r). Firstly, BWA runs much faster for near perfect hits than for hits with many differences, and it stops searching for a hit with l+2 differences if a l-difference hit is found. This means BWA will be very slow if r is high because in this case BWA has to visit hits with many differences and looking for these hits is expensive. Secondly, the alignment algorithm behind makes the speed sensitive to [k log(N)/m], where k is the maximum allowed differences, N the size of database and m the length of a query. In practice, we choose k w.r.t. r and therefore r is the leading factor. I would not recommend to use BWA on data with r>0.02. .PP Pairing is slower for shorter reads. This is mainly because shorter reads have more spurious hits and converting SA coordinates to chromosomal coordinates are very costly. .PP In a practical experiment, BWA is able to map 2 million 32bp reads to a bacterial genome in several minutes, map the same amount of reads to human X chromosome in 8-15 minutes and to the human genome in 15-25 minutes. This result implies that the speed of BWA is insensitive to the size of database and therefore BWA is more efficient when the database is sufficiently large. On smaller genomes, hash based algorithms are usually much faster. .SH NOTES ON LONG-READ ALIGNMENT .PP Command  Charles Plessy committed Jan 20, 2011 442 .B bwasw'  Charles Plessy committed Jan 20, 2011 443 is designed for long-read alignment. The algorithm behind, BWA-SW, is  Charles Plessy committed Jan 20, 2011 444 445 446 similar to BWT-SW, but does not guarantee to find all local hits due to the heuristic acceleration. It tends to be faster and more accurate if the resultant alignment is supported by more seeds, and therefore  Charles Plessy committed Jan 20, 2011 447 BWA-SW usually performs better on long queries than on short ones.  Charles Plessy committed Jan 20, 2011 448   Charles Plessy committed Jan 20, 2011 449 On 350-1000bp reads, BWA-SW is several to tens of times faster than the  Charles Plessy committed Jan 20, 2011 450 existing programs. Its accuracy is comparable to SSAHA2, more accurate  Charles Plessy committed Jan 20, 2011 451 than BLAT. Like BLAT, BWA-SW also finds chimera which may pose a  Charles Plessy committed Jan 20, 2011 452 challenge to SSAHA2. On 10-100kbp queries where chimera detection is  Charles Plessy committed Jan 20, 2011 453 important, BWA-SW is over 10X faster than BLAT while being more  Charles Plessy committed Jan 20, 2011 454 455 sensitive.  Charles Plessy committed Jan 20, 2011 456 BWA-SW can also be used to align ~100bp reads, but it is slower than  Charles Plessy committed Jan 20, 2011 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 the short-read algorithm. Its sensitivity and accuracy is lower than SSAHA2 especially when the sequencing error rate is above 2%. This is the trade-off of the 30X speed up in comparison to SSAHA2's -454 mode. .SH SEE ALSO BWA website , Samtools website .SH AUTHOR Heng Li at the Sanger Institute wrote the key source codes and integrated the following codes for BWT construction: bwtsw , implemented by Chi-Kwong Wong at the University of Hong Kong and IS originally proposed by Nong Ge at the Sun Yat-Sen University and implemented by Yuta Mori. .SH LICENSE AND CITATION .PP The full BWA package is distributed under GPLv3 as it uses source codes from BWT-SW which is covered by GPL. Sorting, hash table, BWT and IS libraries are distributed under the MIT license. .PP If you use the short-read alignment component, please cite the following paper: .PP Li H. and Durbin R. (2009) Fast and accurate short read alignment with  Charles Plessy committed Jan 20, 2011 484 485 486 487 488 489 Burrows-Wheeler transform. Bioinformatics, 25, 1754-60. [PMID: 19451168] .PP If you use the long-read component (BWA-SW), please cite: .PP Li H. and Durbin R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. [PMID: 20080505]  Charles Plessy committed Jan 20, 2011 490 491 492  .SH HISTORY BWA is largely influenced by BWT-SW. It uses source codes from BWT-SW  Charles Plessy committed Jan 20, 2011 493 and mimics its binary file formats; BWA-SW resembles BWT-SW in several  Charles Plessy committed Jan 20, 2011 494 495 496 ways. The initial idea about BWT-based alignment also came from the group who developed BWT-SW. At the same time, BWA is different enough from BWT-SW. The short-read alignment algorithm bears no similarity to  Charles Plessy committed Jan 20, 2011 497 Smith-Waterman algorithm any more. While BWA-SW learns from BWT-SW, it  Charles Plessy committed Jan 20, 2011 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 introduces heuristics that can hardly be applied to the original algorithm. In all, BWA does not guarantee to find all local hits as what BWT-SW is designed to do, but it is much faster than BWT-SW on both short and long query sequences. I started to write the first piece of codes on 24 May 2008 and got the initial stable version on 02 June 2008. During this period, I was acquainted that Professor Tak-Wah Lam, the first author of BWT-SW paper, was collaborating with Beijing Genomics Institute on SOAP2, the successor to SOAP (Short Oligonucleotide Analysis Package). SOAP2 has come out in November 2008. According to the SourceForge download page, the third BWT-based short read aligner, bowtie, was first released in August 2008. At the time of writing this manual, at least three more BWT-based short-read aligners are being implemented.  Charles Plessy committed Jan 20, 2011 513 The BWA-SW algorithm is a new component of BWA. It was conceived in  Charles Plessy committed Jan 20, 2011 514 November 2008 and implemented ten months later.`