Updated 2014 June 8th
I first tried this with BWA. Now I'll try it with Bowtie.
git clone https://github.com/BenLangmead/bowtie.git cd bowtie make
Consider this reference sequence, which is the sequence "ACGTACGTACGTACGTAGGTACGTAGGG" repeated 20 times:
>artificial
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
ACGTACGTACGTACGTAGGTACGTAGGG
and this read:
>tag
ACGTACGTACGTACGTAGGTACGTA
The next steps are to build an index and then align our read to the index:
#build index bowtie-build ref.fa ref #if you want to scroll up and down the usage #bowtie 2>&1 | less #here are what the parameters mean #-k <int> report up to <int> good alignments per read (default: 1) #-f query input files are (multi-)FASTA .fa/.mfa #-S/--sam write hits in SAM format bowtie -k 40 -f -S ref read.fa @HD VN:1.0 SO:unsorted @SQ SN:artificial LN:560 @PG ID:Bowtie VN:1.0.1 CL:"bowtie -k 40 -f -S ref read.fa" tag 0 artificial 29 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 57 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 85 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 113 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 141 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 169 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 197 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 225 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 253 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 281 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 309 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 337 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 365 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 393 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 421 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 449 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 477 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 505 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 533 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 1 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 289 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 261 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 233 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 205 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 177 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 149 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 121 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 93 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 65 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 37 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 9 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 513 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 485 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 457 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 429 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 401 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 373 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 345 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 tag 0 artificial 317 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:2 MD:Z:9G9G5 NM:i:2 # reads processed: 1 # reads with at least one reported alignment: 1 (100.00%) # reads that failed to align: 0 (0.00%) Reported 39 alignments to 1 output stream(s)
To report all the best alignments:
bowtie -f -a -S --best --strata ref read.fa @HD VN:1.0 SO:unsorted @SQ SN:artificial LN:560 @PG ID:Bowtie VN:1.0.1 CL:"bowtie -f -a -S --best --strata ref read.fa" tag 0 artificial 505 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 477 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 449 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 421 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 393 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 365 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 337 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 309 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 281 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 253 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 225 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 197 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 169 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 141 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 113 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 85 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 57 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 29 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 1 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 tag 0 artificial 533 255 25M * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XA:i:0 MD:Z:25 NM:i:0 # reads processed: 1 # reads with at least one reported alignment: 1 (100.00%) # reads that failed to align: 0 (0.00%) Reported 20 alignments to 1 output stream(s)
If I want to exclude tags mapping to m places:
bowtie -f -a -m 19 -S --best --strata ref read.fa @HD VN:1.0 SO:unsorted @SQ SN:artificial LN:560 @PG ID:Bowtie VN:1.0.1 CL:"bowtie -f -a -m 19 -S --best --strata ref read.fa" tag 4 * 0 0 * * 0 0 ACGTACGTACGTACGTAGGTACGTA IIIIIIIIIIIIIIIIIIIIIIIII XM:i:19 # reads processed: 1 # reads with at least one reported alignment: 0 (0.00%) # reads that failed to align: 0 (0.00%) # reads with alignments suppressed due to -m: 1 (100.00%) No alignments
I could not find an option for reporting all alignments in BWA (which may exist?); the default behaviour for BWA is to report one random location for multimapping tags. Using the -a -m 10 -S --best --strata parameters, does exactly what I want; report all alignments but keep only the best hits, however if a tag maps to more than 10 places mark it as unmapped.
See the Bowtie manual for more information and usage examples.
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