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blasr - Online in the Cloud

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PROGRAM:

NAME


blasr - Map SMRT Sequences to a reference genome.

SYNOPSIS


blasr reads.bam genome.fasta -bam -out out.bam

blasr reads.fasta genome.fasta

blasr reads.fasta genome.fasta -sa genome.fasta.sa

blasr reads.bax.h5 genome.fasta [-sa genome.fasta.sa]

blasr reads.bax.h5 genome.fasta -sa genome.fasta.sa -maxScore -100 -minMatch 15 ...

blasr reads.bax.h5 genome.fasta -sa genome.fasta.sa -nproc 24 -out alignment.out ...

DESCRIPTION


blasr is a read mapping program that maps reads to positions in a genome by clustering
short exact matches between the read and the genome, and scoring clusters using alignment.
The matches are generated by searching all suffixes of a read against the genome using a
suffix array. Global chaining methods are used to score clusters of matches.

The only required inputs to blasr are a file of reads and a reference genome. It is
exremely useful to have read filtering information, and mapping runtime may decrease
substantially when a precomputed suffix array index on the reference sequence is
specified.

Although reads may be input in FASTA format, the recommended input is PacBio BAM files
because these contain qualtiy value information that is used in the alignment and produces
higher quality variant detection. Although alignments can be output in various formats,
the recommended output format is PacBio BAM. Support for bax.h5 and plx.h5 files will be
DEPRECATED. Support for region tables for h5 files will be DEPRECATED.

When suffix array index of a genome is not specified, the suffix array is built before
producing alignment. This may be prohibitively slow when the genome is large (e.g. Human).
It is best to precompute the suffix array of a genome using the program sawriter(1), and
then specify the suffix array on the command line using -sa genome.fa.sa.

The optional parameters are roughly divided into three categories: control over anchoring,
alignment scoring, and output.

The default anchoring parameters are optimal for small genomes and samples with up to 5%
divergence from the reference genome. The main parameter governing speed and sensitivity
is the -minMatch parameter. For human genome alignments, a value of 11 or higher is
recommended. Several methods may be used to speed up alignments, at the expense of
possibly decreasing sensitivity.

Regions that are too repetitive may be ignored during mapping by limiting the number of
positions a read maps to with the -maxAnchorsPerPosition option. Values between 500 and
1000 are effective in the human genome.

For small genomes such as bacterial genomes or BACs, the default parameters are sufficient
for maximal sensitivity and good speed.

OPTIONS


Input Files

Reads

reads.bam
A PacBio BAM file of reads. This is the preferred input to blasr
because rich quality value (insertion,deletion, and substitution
quality values) information is maintained. The extra quality
information improves variant detection and mapping speed.

reads.fasta
A multi-fasta file of reads, though any fasta file is valid input

reads.bax.h5|reads.plx.h5
the old DEPRECATED output format of SMRT reads.

input.fofn
File of file names

-sa suffixArrayFile
Use the suffix array 'sa' for detecting matches between the reads and the
reference. The suffix array has been prepared by the sawriter(1) program.

-ctab tab
A table of tuple counts used to estimate match significance. This is by the
program 'printTupleCountTable'. While it is quick to generate on the fly,
if there are many invocations of blasr, it is useful to precompute the ctab.

-regionTable table (DEPRECATED)
Read in a read-region table in HDF format for masking portions of reads.
This may be a single table if there is just one input file, or a fofn. When
a region table is specified, any region table inside the reads.plx.h5 or
reads.bax.h5 files are ignored.
(DEPRECATED) Options for modifying reads.

There is ancilliary information about substrings of reads that is stored in a
'region table' for each read file. Because HDF is used, the region table may be
part of the .bax.h5 or .plx.h5 file, or a separate file. A contiguously read
substring from the template is a subread, and any read may contain multiple
subreads. The boundaries of the subreads may be inferred from the region table
either directly or by definition of adapter boundaries. Typically region tables
also contain information for the location of the high and low quality regions of
reads. Reads produced by spurious reads from empty ZMWs have a high quality start
coordinate equal to high quality end, making no usable read.

-useccs
Align the circular consensus sequence (ccs), then report alignments of the
ccs subreads to the window that the ccs was mapped to. Only alignments of
the subreads are reported.

-useccsall
Similar to -useccs, except all subreads are aligned, rather than just the
subreads used to call the ccs. This will include reads that only cover part
of the template.

-useccsdenovo
Align the circular consensus, and report only the alignment of the ccs
sequence.

-noSplitSubreads (false)
Do not split subreads at adapters. This is typically only useful when the
genome in an unrolled version of a known template, and contains template-
adapter-reverse_template sequence.

-ignoreRegions (false)
Ignore any information in the region table.

-ignoreHQRegions (false)
Ignore any hq regions in the region table.
Alignments To Report

-bestn n (10)
Report the top n alignments.

-hitPolicy (all)
Specify a policy to treat multiple hits from [all, allbest, random,
randombest, leftmost]

all report all alignments.

allbest
report all equally top scoring alignments.

random report a random alignment.

randombest
report a random alignment from multiple equally top scoring
alignments.

leftmost
report an alignment which has the best alignmentscore and has the
smallest mapping coordinate in any reference.

-placeRepeatsRandomly (false)
DEPRECATED! If true, equivalent to -hitPolicy randombest.

-randomSeed (0)
Seed for random number generator. By default (0), use current time as seed.

-noSortRefinedAlignments (false)
Once candidate alignments are generated and scored via sparse dynamic
programming, they are rescored using local alignment that accounts for
different error profiles. Resorting based on the local alignment may change
the order the hits are returned.

-allowAdjacentIndels
When specified, adjacent insertion or deletions are allowed. Otherwise,
adjacent insertion and deletions are merged into one operation. Using
quality values to guide pairwise alignments may dictate that the higher
probability alignment contains adjacent insertions or deletions. Current
tools such as GATK do not permit this and so they are not reported by
default.
Output Formats and Files

-out out (terminal)
Write output to out.

-sam Write output in SAM format.

-m t If not printing SAM, modify the output of the alignment.

When t is:

0 Print blast like output with |'s connecting matched nucleotides.

1 Print only a summary: score and pos.

2 Print in Compare.xml format.

3 Print in vulgar format (DEPRECATED).

4 Print a longer tabular version of the alignment.

5 Print in a machine-parsable format that is read by
compareSequences.py.

-header
Print a header as the first line of the output file describing the contents
of each column.

-titleTable tab (NULL)
Construct a table of reference sequence titles. The reference sequences are
enumerated by row, 0,1,... The reference index is printed in alignment
results rather than the full reference name. This makes output concise,
particularly whenvery verbose titles exist in reference names.

-unaligned file
Output reads that are not aligned to file

-clipping [none|hard|subread|soft] (none)

Use no/hard/subread/soft clipping, ONLY for SAM/BAM output.

-printSAMQV (false)
Print quality values to SAM output.

-cigarUseSeqMatch (false)
CIGAR strings in SAM/BAM output use '=' and 'X' to represent sequence match
and mismatch instead of 'M'.
Options for anchoring alignment regions.

This will have the greatest effect on speed and sensitivity.

-minMatch m (12)
Minimum seed length. Higher minMatch will speed up alignment, but decrease
sensitivity.

-maxMatch l (inf)
Stop mapping a read to the genome when the lcp length reaches l. This is
useful when the query is part of the reference, for example when
constructing pairwise alignments for de novo assembly.

-maxLCPLength l (inf)
The same as -maxMatch.

-maxAnchorsPerPosition m (10000)
Do not add anchors from a position if it matches to more than m locations in
the target.

-advanceExactMatches E (0)
Another trick for speeding up alignments with match - E fewer anchors.
Rather than finding anchors between the read and the genome at every
position in the read, when an anchor is found at position i in a read of
length L, the next position in a read to find an anchor is at i+L-E. Use
this when alignining already assembled contigs.

-nCandidates n (10)
Keep up to n candidates for the best alignment. A large value of n will
slow mapping because the slower dynamic programming steps are applied to
more clusters of anchors which can be a rate limiting step when reads are
very long.

-concordant (false)
Map all subreads of a zmw (hole) to where the longest full pass subread of
the zmw aligned to. This requires to use the region table and hq regions.
This option only works when reads are in base or pulse h5 format.

-concordantTemplate (mediansubread)
Select a full pass subread of a zmw as template for concordant mapping.
longestsubread - use the longest full pass subread mediansubread - use the
median length full pass subread typicalsubread - use the second longest full
pass subread if length of the longest full pass subread is an outlier

-fastMaxInterval (false)
Fast search maximum increasing intervals as alignment candidates. The search
is not as exhaustive as the default, but is much faster.

-aggressiveIntervalCut (false)
Agreesively filter out non-promising alignment candidates, if there exists
at least one promising candidate. If this option is turned on, blasr is
likely to ignore short alignments of ALU elements.

-fastSDP (false)
Use a fast heuristic algorithm to speed up sparse dynamic programming.
Options for Refining Hits

-sdpTupleSize K (11)
Use matches of length K to speed dynamic programming alignments. This
controls accuracy of assigning gaps in pairwise alignments once a mapping
has been found, rather than mapping sensitivity itself.

-scoreMatrix score matrix string
Specify an alternative score matrix for scoring fasta reads. The matrix is
in the format

A C G T N
A a b c d e
C f g h i j
G k l m n o
T p q r s t
N u v w x y

The values a...y should be input as a quoted space separated string: "a b c
... y". Lowerf scores are better, so matches should be less than mismatches
e.g. a,g,m,s = -5 (match), mismatch = 6.

-affineOpen value (10)
Set the penalty for opening an affine alignment.

-affineExtend a (0)
Change affine (extension) gap penalty. Lower value allows more gaps.
Options for overlap/dynamic programming alignments and pairwise overlap for de novo
assembly.

-useQuality (false)
Use substitution/insertion/deletion/merge quality values to score gap and
mismatch penalties in pairwise alignments. Because the insertion and
deletion rates are much higher than substitution, this will make many
alignments favor an insertion/deletion over a substitution.nNaive consensus
calling methods will then often miss substitution polymorphisms. This option
should be used when calling consensus using the Quiver method. Furthermore,
when not using quality values to score alignments, there will be a lower
consensus accuracy in homolymer regions.

-affineAlign (false)
Refine alignment using affine guided align.
Options for filtering reads and alignments

-minReadLength l (50)
Skip reads that have a full length less than l. Subreads may be shorter.

-minSubreadLength l (0)
Do not align subreads of length less than l.

-minRawSubreadScore m (0)
Do not align subreads whose quality score in region table is less than m
(quality scores should be in range [0, 1000]).

-maxScore m (-200)
Maximum score to output (high is bad, negative good).

-minAlnLength
(0) Report alignments only if their lengths are greater than minAlnLength.

-minPctSimilarity (0) Report alignments only if their percentage similairty is
greater than minPctSimilarity.

-minPctAccuracy
(0) Report alignments only if their percentage accuray is greater than
minAccuracy.
Options for parallel alignment

-nproc N (1)
Align using N processes. All large data structures such as the suffix array
and tuple count table are shared.

-start S (0)
Index of the first read to begin aligning. This is useful when multiple
instances are running on the same data, for example when on a multi-rack
cluster.

-stride S (1)
Align one read every S reads.
Options for subsampling reads.

-subsample (0)
Proportion of reads to randomly subsample (expressed as a decimal) and
align.

-holeNumbers LIST
When specified, only align reads whose ZMW hole numbers are in LIST. LIST
is a comma-delimited string of ranges, such as '1,2,3,10-13'. This option
only works when reads are in bam, bax.h5 or plx.h5 format.

-h Print help information.

CITATION


To cite BLASR, please use: Chaisson M.J., and Tesler G., Mapping single molecule
sequencing reads using Basic Local Alignment with Successive Refinement (BLASR): Theory
and Application, BMC Bioinformatics 2012, 13:238.

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