What it does

BFAST facilitates the fast and accurate mapping of short reads to reference sequences. Some advantages of BFAST include: * Speed: enables billions of short reads to be mapped quickly. * Accuracy: A priori probabilities for mapping reads with defined set of variants * An easy way to measurably tune accuracy at the expense of speed. Specifically, BFAST was designed to facilitate whole-genome resequencing, where mapping billions of short reads with variants is of utmost importance.

BFAST supports both Illumina and ABI SOLiD data, as well as any other Next-Generation Sequencing Technology (454, Helicos), with particular emphasis on sensitivity towards errors, SNPs and especially indels. Other algorithms take short-cuts by ignoring errors, certain types of variants (indels), and even require further alignment, all to be the "fastest" (but still not complete). BFAST is able to be tuned to find variants regardless of the error-rate, polymorphism rate, or other factors.

Please cite the website "http://bfast.sourceforge.net" as well as the accompanying papers:

Homer N, Merriman B, Nelson SF. BFAST: An alignment tool for large scale genome resequencing. PMID: 19907642 PLoS ONE. 2009 4(11): e7767. http://dx.doi.org/10.1371/journal.pone.0007767

Homer N, Merriman B, Nelson SF. Local alignment of two-base encoded DNA sequence. BMC Bioinformatics. 2009 Jun 9;10(1):175. PMID: 19508732 http://dx.doi.org/10.1186/1471-2105-10-175

Know what you are doing

There is no such thing (yet) as an automated gearshift in short read mapping. It is all like stick-shift driving in San Francisco. In other words = running this tool with default parameters will probably not give you meaningful results. A way to deal with this is to understand the parameters by carefully reading the documentation and experimenting. Fortunately, Galaxy makes experimenting easy.

Input formats

BFAST accepts files in Sanger FASTQ format. Use the FASTQ Groomer to prepare your files.

Outputs

The output is in SAM format, and has the following columns:

  Column  Description
--------  --------------------------------------------------------
1  QNAME  Query (pair) NAME
2  FLAG   bitwise FLAG
3  RNAME  Reference sequence NAME
4  POS    1-based leftmost POSition/coordinate of clipped sequence
5  MAPQ   MAPping Quality (Phred-scaled)
6  CIGAR  extended CIGAR string
7  MRNM   Mate Reference sequence NaMe ('=' if same as RNAME)
8  MPOS   1-based Mate POSition
9  ISIZE  Inferred insert SIZE
10 SEQ    query SEQuence on the same strand as the reference
11 QUAL   query QUALity (ASCII-33 gives the Phred base quality)
12 OPT    variable OPTional fields in the format TAG:VTYPE:VALU

The flags are as follows:

Flag  Description
------  -------------------------------------
0x0001  the read is paired in sequencing
0x0002  the read is mapped in a proper pair
0x0004  the query sequence itself is unmapped
0x0008  the mate is unmapped
0x0010  strand of the query (1 for reverse)
0x0020  strand of the mate
0x0040  the read is the first read in a pair
0x0080  the read is the second read in a pair
0x0100  the alignment is not primary

It looks like this (scroll sideways to see the entire example):

QNAME  FLAG  RNAME  POS  MAPQ  CIAGR  MRNM  MPOS  ISIZE  SEQ  QUAL  OPT
HWI-EAS91_1_30788AAXX:1:1:1761:343  4  *  0  0  *  *  0  0  AAAAAAANNAAAAAAAAAAAAAAAAAAAAAAAAAAACNNANNGAGTNGNNNNNNNGCTTCCCACAGNNCTGG  hhhhhhh;;hhhhhhhhhhh^hOhhhhghhhfhhhgh;;h;;hhhh;h;;;;;;;hhhhhhghhhh;;Phhh
HWI-EAS91_1_30788AAXX:1:1:1578:331  4  *  0  0  *  *  0  0  GTATAGANNAATAAGAAAAAAAAAAATGAAGACTTTCNNANNTCTGNANNNNNNNTCTTTTTTCAGNNGTAG  hhhhhhh;;hhhhhhhhhhhhhhhhhhhhhhhhhhhh;;h;;hhhh;h;;;;;;;hhhhhhhhhhh;;hhVh

BFAST settings

All of the options have a default value. You can change any of them. Most of the options in BFAST have been implemented here.

BFAST parameter list

This is an exhaustive list of BFAST options:

For match:

-o  STRING   Specifies the offset [Use all]
-l      Specifies to load all main or secondary indexes into memory
-A  INT    0: NT space 1: Color space [0]
-k  INT    Specifies to truncate all indexes to have the given key size
(must be greater than the hash width) [Not Using]
-K  INT    Specifies the maximum number of matches to allow before a key
is ignored [8]
-M  INT    Specifies the maximum total number of matches to consider
before the read is discarded [384]
-w  INT    0: consider both strands 1: forward strand only 2: reverse
strand only [0]
-n  INT   Specifies the number of threads to use [1]
-t         Specifies to output timing information

For localalign:

-x  FILE  Specifies the file name storing the scoring matrix
-u        Do ungapped local alignment (the default is gapped).
-U         Do not use mask constraints from the match step
-A  INT    0: NT space 1: Color space [0]
-o  INT    Specifies the number of bases before and after the match to
include in the reference genome
-M  INT    Specifies the maximum total number of matches to consider
before the read is discarded [384]
-q  INT    Specifies the average mismatch quality
-n  INT   Specifies the number of threads to use [1]
-t         Specifies to output timing information

For postprocess:

-a  INT    Specifies the algorithm to choose the alignment for each end of the read:

  0: No filtering will occur.
  1: All alignments that pass the filters will be output
  2: Only consider reads that have been aligned uniquely
  3: Choose uniquely the alignment with the best score
  4: Choose all alignments with the best score

-A  INT    0: NT space 1: Color space [0]
-U      Specifies that pairing should not be performed
-R          Specifies that paired reads are on opposite strands
-q   INT    Specifies the average mismatch quality
-x  FILE  Specifies the file name storing the scoring matrix
-z          Specifies to output a random best scoring alignment (with -a 3)
-r   FILE  Specifies to add the RG in the specified file to the SAM
header and updates the RG tag (and LB/PU tags if present) in
the reads (SAM only)
-n  INT   Specifies the number of threads to use [1]
-t         Specifies to output timing information