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279 lines
10 KiB
Groff
279 lines
10 KiB
Groff
.TH SPIDEY 1 2005-01-25 NCBI "NCBI Tools User's Manual"
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.SH NAME
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spidey \- align mRNA sequences to a genome
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.SH SYNOPSIS
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.B spidey
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[\|\fB\-\fP\|]
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[\|\fB\-F\fP\ \fIN\fP\|]
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[\|\fB\-G\fP\|]
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[\|\fB\-L\fP\ \fIN\fP\|]
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[\|\fB\-M\fP\ \fIfilename\fP\|]
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[\|\fB\-N\fP\ \fIfilename\fP\|]
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[\|\fB\-R\fP\ \fIfilename\fP\|]
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[\|\fB\-S\fP\ \fIp/m\fP\|]
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[\|\fB\-T\fP\ \fIN\fP\|]
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[\|\fB\-X\fP\|]
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[\|\fB\-a\fP\ \fIfilename\fP\|]
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[\|\fB\-c\fP\ \fIN\fP\|]
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[\|\fB\-d\fP\|]
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[\|\fB\-e\fP\ \fIX\fP\|]
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[\|\fB\-f\fP\ \fIX\fP\|]
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[\|\fB\-g\fP\ \fIX\fP\|]
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\fB\-i\fP\ \fIfilename\fP
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[\|\fB\-j\fP\|]
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[\|\fB\-k\fP\ \fIfilename\fP\|]
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[\|\fB\-l\fP\ \fIN\fP\|]
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\fB\-m\fP\ \fIfilename\fP
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[\|\fB\-n\fP\ \fIN\fP\|]
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[\|\fB\-o\fP\ \fIstr\fP\|]
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[\|\fB\-p\fP\ \fIN\fP\|]
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[\|\fB\-r\fP\ \fIc/d/m/p/v\fP\|]
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[\|\fB\-s\fP\|]
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[\|\fB\-t\fP\ \fIfilename\fP\|]
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[\|\fB\-u\fP\|]
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[\|\fB\-w\fP\|]
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.SH DESCRIPTION
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\fBspidey\fP is a tool for aligning one or more mRNA sequences to a
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given genomic sequence. \fBspidey\fP was written with two main goals
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in mind: find good alignments regardless of intron size; and avoid
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getting confused by nearby pseudogenes and paralogs. Towards the
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first goal, \fBspidey\fP uses BLAST and Dot View (another local
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alignment tool) to find its alignments; since these are both local
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alignment tools, \fBspidey\fP does not intrinsically favor shorter or
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longer introns and has no maximum intron size. To avoid mistakenly
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including exons from paralogs and pseudogenes, \fBspidey\fP first
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defines windows on the genomic sequence and then performs the
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mRNA-to-genomic alignment separately within each window. Because of
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the way the windows are constructed, neighboring paralogs or
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pseudogenes should be in separate windows and should not be included
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in the final spliced alignment.
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.SS Initial alignments and construction of genomic windows
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\fBspidey\fP takes as input a single genomic sequence and a set of
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mRNA accessions or FASTA sequences. All processing is done one mRNA
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sequence at a time. The first step for each mRNA sequence is a
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high-stringency BLAST against the genomic sequence. The resulting
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hits are analyzed to find the genomic windows.
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.PP
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The BLAST alignments are sorted by score and then assigned into
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windows by a recursive function which takes the first alignment and
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then goes down the alignment list to find all alignments that are
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consistent with the first (same strand of mRNA, both the mRNA and
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genomic coordinates are nonoverlapping and linearly consistent). On
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subsequent passes, the remaining alignments are examined and are put
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into their own nonoverlapping, consistent windows, until no alignments
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are left. Depending on how many gene models are desired, the
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top \fIn\fP windows are chosen to go on to the next step and the others
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are deleted.
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.SS Aligning in each window
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Once the genomic windows are constructed, the initial BLAST alignments
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are freed and another BLAST search is performed, this time with the
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entire mRNA against the genomic region defined by the window, and at a
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lower stringency than the initial search. \fBspidey\fP then uses a
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greedy algorithm to generate a high-scoring, nonoverlapping subset of
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the alignments from the second BLAST search. This consistent set is
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analyzed carefully to make sure that the entire mRNA sequence is
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covered by the alignments. When gaps are found between the
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alignments, the appropriate region of genomic sequence is searched
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against the missing mRNA, first using a very low-stringency BLAST and,
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if the BLAST fails to find a hit, using DotView functions to locate
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the alignment. When gaps are found at the ends of the alignments, the
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BLAST and DotView searches are actually allowed to extend past the
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boundaries of the window. If the 3' end of the mRNA does not align
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completely, it is first examined for the presence of a poly(A) tail.
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No attempt is made to align the portion of the mRNA that seems to be a
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poly(A) tail; sometimes there is a poly(A) tail that does align to the
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genomic sequence, and these are noted because they indicate the
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possibility of a pseudogene.
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.PP
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Now that the mRNA is completely covered by the set of alignments, the
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boundaries of the alignments (there should be one alignment per exon
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now) are adjusted so that the alignments abut each other precisely and
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so that they are adjacent to good splice donor and acceptor sites.
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Most commonly, two adjacent exons' alignments overlap by as much as 20
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or 30 base pairs on the mRNA sequence. The true exon boundary may lie
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anywhere within this overlap, or (as we have seen empirically) even a
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few base pairs outside the overlap. To position the exon boundaries,
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the overlap plus a few base pairs on each side is examined for splice
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donor sites, using functions that have different splice matrices
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depending on the organism chosen. The top few splice donor sites (by
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score) are then evaluated as to how much they affect the original
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alignment boundaries. The site that affects the boundaries the least
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is chosen, and is evaluated as to the presence of an acceptor site.
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The alignments are truncated or extended as necessary so that they
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terminate at the splice donor site and so that they do not overlap.
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.SS Final result
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The windows are examined carefully to get the percent identity per
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exon, the number of gaps per exon, the overall percent identity, the
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percent coverage of the mRNA, presence of an aligning or non-aligning
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poly(A) tail, number of splice donor sites and the presence or absence
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of splice donor and acceptor sites for each exon, and the occurrence
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of an mRNA that has a 5' or 3' end (or both) that does not align to
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the genomic sequence. If the overall percent identity and percent
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length coverage are above the user-defined cutoffs, a summary report
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is printed, and, if requested, a text alignment showing identities and
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mismatches is also printed.
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.SS Interspecies alignments
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\fBspidey\fP is capable of performing interspecies alignments. The
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major difference in interspecies alignments is that the mRNA-genomic
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identity will not be close to 100% as it is in intraspecies
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alignments; also, the alignments have numerous and lengthy gaps. If
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\fBspidey\fP is used in its normal mode to do interspecies alignments,
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it produces gene models with many, many short exons. When the
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interspecies flag is set, \fBspidey\fP uses different BLAST parameters
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to encourage longer and more gaps and to not penalize as heavily for
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mismatches. This way, the alignments for the exons are much longer
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and more closely approximate the actual gene structure.
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.SS Extracting CDS alignments
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When \fBspidey\fP is run in network-aware mode or when ASN.1 files are
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used for the mRNA records, it is capable of extracting a CDS alignment
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from an mRNA alignment and printing the CDS information also. Since
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the CDS alignment is just a subset of the mRNA alignment, it is
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relatively straightforward to truncate the exon alignments as
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necessary and to generate a CDS alignment. Furthermore, the
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untranslated regions are now defined, so the percent identity for the
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5' and 3' untranslated regions is also calculated.
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.PP
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.SH OPTIONS
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A summary of options is included below.
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.TP
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\fB\-\fP
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Print usage message.
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.TP
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\fB\-F\fP\ \fIN\fP
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Start of genomic interval desired (from; 0-based).
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.TP
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\fB\-G\fP
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Input file is a GI list.
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.TP
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\fB\-L\fP\ \fIN\fP
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The extra-large intron size to use (default = 220000).
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.TP
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\fB\-M\fP\ \fIfilename\fP
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File with donor splice matrix.
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.TP
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\fB\-N\fP\ \fIfilename\fP
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File with acceptor splice matrix.
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.TP
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\fB\-R\fP\ \fIfilename\fP
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File (including path) to repeat blast database for filtering.
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.TP
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\fB\-S\fP\ \fIp/m\fP
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Restrict to plus (p) or minus (m) strand of genomic sequence.
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.TP
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\fB\-T\fP\ \fIN\fP
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Stop of genomic interval desired (to; 0-based).
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.TP
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\fB\-X\fP
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Use extra-large intron sizes (increases the limit for initial and
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terminal introns from 100kb to 240kb and for all others from 35kb to
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120kb); may result in significantly longer compute times.
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.TP
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\fB\-a\fP\ \fIfilename\fP
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Output file for alignments when directed to a separate file with
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\fB-p\ 3\fP (default = spidey.aln).
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.TP
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\fB\-c\fP\ \fIN\fP
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Identity cutoff, in percent, for quality control purposes.
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.TP
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\fB\-d\fP
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Also try to align coding sequences corresponding to the given mRNA
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records (may require network access).
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.TP
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\fB\-e\fP\ \fIX\fP
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First-pass e-value (default = 1.0e-10). Higher values increase speed
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at the cost of sensitivity.
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.TP
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\fB\-f\fP\ \fIX\fP
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Second-pass e-value (default = 0.001).
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.TP
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\fB\-g\fP\ \fIX\fP
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Third-pass e-value (default = 10).
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.TP
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\fB\-i\fP\ \fIfilename\fP
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Input file containing the genomic sequence in ASN.1 or FASTA format.
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If your computer is running on a network that can access GenBank, you
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can substitute the desired accession number for the filename.
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.TP
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\fB\-j\fP
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Print ASN.1 alignment?
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.TP
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\fB\-k\fP\ \fIfilename\fP
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File for ASN.1 output with \fB-k\fP (default = spidey.asn).
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.TP
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\fB\-l\fP\ \fIN\fP
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Length coverage cutoff, in percent.
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.TP
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\fB\-m\fP\ \fIfilename\fP
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Input file containing the mRNA sequence(s) in ASN.1 or FASTA format,
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or a list of their accessions (with \fB-G\fP). If your computer is
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running on a network that can access GenBank, you can substitute a
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single accession number for the filename.
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.TP
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\fB\-n\fP\ \fIN\fP
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Number of gene models to return per input mRNA (default = 1).
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.TP
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\fB\-o\fP\ \fIstr\fP
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Main output file (default = stdout; contents controlled by \fB-p\fP).
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.TP
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\fB\-p\fP\ \fIN\fP
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Print alignment?
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.RS
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.PD 0
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.IP \fB0\fP
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summary and alignments together (default)
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.IP \fB1\fP
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just the summary
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.IP \fB2\fP
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just the alignments
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.IP \fB3\fP
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summary and alignments in different files
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.PD
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.RE
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.TP
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\fB\-r\fP\ \fIc/d/m/p/v\fP
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Organism of genomic sequence, used to determine splice matrices.
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.RS
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.PD 0
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.IP \fBc\fP
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C. elegans
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.IP \fBd\fP
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Drosophila
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.IP \fBm\fP
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Dictyostelium discoideum
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.IP \fBp\fP
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plant
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.IP \fBv\fP
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vertebrate (default)
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.PD
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.RE
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.TP
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\fB\-s\fP
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Tune for interspecies alignments.
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.TP
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\fB\-t\fP\ \fIfilename\fP
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File with feature table, in 4 tab-delimited columns:
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.RS
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.PD 0
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.IP \fIseqid\fP
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(e.g., \fBNM_04377.1\fP)
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.IP \fIname\fP
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(only \fBrepetitive_region\fP is currently supported)
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.IP \fIstart\fP
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(0-based)
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.IP \fIstop\fP
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(0-based)
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.PD
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.RE
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.TP
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\fB\-u\fP
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Make a multiple alignment of all input mRNAs (which must overlap on
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the genomic sequence).
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.TP
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\fB\-w\fP
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Consider lowercase characters in input FASTA sequences to be masked.
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.SH AUTHOR
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Sarah Wheelan and others at the National Center for Biotechnology
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Information; Steffen Moeller contributed to this documentation.
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.SH SEE ALSO
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.BR blast (1),
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<http://www.ncbi.nlm.nih.gov/spidey>
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