formats.tex

hmmer源程序

between the sequence lines in a block. Each block in a multi-block
file of a long alignment must have its sequences in the same order in
each block. The names are checked to verify that this is the case; if
not, only a warning is generated. (In manually constructed files, some
users may wish to use shorthand names in subsequent blocks after an
initial block with full names -- but this isn't recommended.)
\end{enumerate}

\section{Stockholm, the recommended multiple sequence alignment format}

While we recommend a community standard format (FASTA) for unaligned
sequence files, the recommended multiple alignment file format is not
a community standard.  The Pfam Consortium developed a format (based
on extended SELEX) called ``Stockholm format''.  The reasons for this
are two-fold. First, there really is no standard accepted format for
multiple sequence alignment files, so we don't feel guilty about
inventing a new one. Second, the formats of popular multiple alignment
software (e.g. CLUSTAL, GCG MSF, PHYLIP) do not support rich
documentation and markup of the alignment.  Stockholm format was
developed to support extensible markup of multiple sequence
alignments, and we use this capability extensively in both RNA work
(with structural markup) and the Pfam database (with extensive use of
both annotation and markup).

\subsection{A minimal Stockholm file}
\begin{verbatim}
# STOCKHOLM 1.0

seq1  ACDEF...GHIKL
seq2  ACDEF...GHIKL
seq3  ...EFMNRGHIKL

seq1  MNPQTVWY
seq2  MNPQTVWY
seq3  MNPQT...

\end{verbatim}

The simplest Stockholm file is pretty intuitive, easily generated in a
text editor. It is usually easy to convert alignment formats into a
``least common denominator'' Stockholm format. For instance, SELEX,
GCG's MSF format, and the output of the CLUSTALV multiple alignment
program are all similar interleaved formats.

The first line in the file must be \verb+# STOCKHOLM 1.x+, where
\verb+x+ is a minor version number for the format specification
(and which currently has no effect on my parsers). This line allows a
parser to instantly identify the file format.

In the alignment, each line contains a name, followed by the aligned
sequence. A dash or period denotes a gap. If the alignment is too long
to fit on one line, the alignment may be split into multiple blocks,
with blocks separated by blank lines. The number of sequences, their
order, and their names must be the same in every block. Within a given
block, each (sub)sequence (and any associated \verb+#=GR+ and
\verb+#=GC+ markup, see below) is of equal length, called the
\textit{block length}. Block lengths may differ from block to block;
the block length must be at least one residue, and there is no
maximum.  

Other blank lines are ignored. You can add comments to the file on
lines starting with a \verb+#+.

All other annotation is added using a tag/value comment style. The
tag/value format is inherently extensible, and readily made
backwards-compatible; unrecognized tags will simply be ignored. Extra
annotation includes consensus and individual RNA or protein secondary
structure, sequence weights, a reference coordinate system for the
columns, and database source information including name, accession
number, and coordinates (for subsequences extracted from a longer
source sequence) See below for details.

\subsection{Syntax of Stockholm markup}

There are four types of Stockholm markup annotation, for per-file,
per-sequence, per-column, and per-residue annotation:

\begin{wideitem}
\item {\emprog{#=GF <tag> <s>}}
	Per-file annotation. \prog{<s>} is a free format text line
	of annotation type \prog{<tag>}. For example, \prog{#=GF DATE
	April 1, 2000}. Can occur anywhere in the file, but usually
	all the \prog{#=GF} markups occur in a header.

\item {\emprog{#=GS <seqname> <tag> <s>}}
	Per-sequence annotation. \prog{<s>} is a free format text line
	of annotation type \prog{tag} associated with the sequence
	named \prog{<seqname>}. For example, \prog{#=GS seq1
	SPECIES_SOURCE Caenorhabditis elegans}. Can occur anywhere
	in the file, but in single-block formats (e.g. the Pfam
	distribution) will typically follow on the line after the
	sequence itself, and in multi-block formats (e.g. HMMER
	output), will typically occur in the header preceding the
	alignment but following the \prog{#=GF} annotation.

\item {\emprog{#=GC <tag> <...s...>}
	Per-column annotation. \prog{<...s...>} is an aligned text line
	of annotation type \prog{<tag>}.
        \verb+#=GC+ lines are
	associated with a sequence alignment block; \prog{<...s...>}
	is aligned to the residues in the alignment block, and has
	the same length as the rest of the block.
	Typically \verb+#=GC+ lines are placed at the end of each block.

\item {\emprog{#=GR <seqname> <tag> <.....s.....>}
	Per-residue annotation. \prog{<...s...>} is an aligned text line
	of annotation type \prog{<tag>}, associated with the sequence
	named \prog{<seqname>}. 
	\verb+#=GR+ lines are 
	associated with one sequence in a sequence alignment block; 
	\prog{<...s...>}
	is aligned to the residues in that sequence, and has
	the same length as the rest of the block.
	Typically
        \verb+#=GR+ lines are placed immediately following the
	aligned	sequence they annotate.
\end{wideitem}

\subsection{Semantics of Stockholm markup}

Any Stockholm parser will accept syntactically correct files, but is
not obligated to do anything with the markup lines. It is up to the
application whether it will attempt to interpret the meaning (the
semantics) of the markup in a useful way. At the two extremes are the
Belvu alignment viewer and the HMMER profile hidden Markov model
software package.

Belvu simply reads Stockholm markup and displays it, without trying to
interpret it at all. The tag types (\prog{#=GF}, etc.) are sufficient
to tell Belvu how to display the markup: whether it is attached to the
whole file, sequences, columns, or residues.

HMMER uses Stockholm markup to pick up a variety of information from
the Pfam multiple alignment database. The Pfam consortium therefore
agrees on additional syntax for certain tag types, so HMMER can parse
some markups for useful information. This additional syntax is imposed
by Pfam, HMMER, and other software of mine, not by Stockholm format
per se. You can think of Stockholm as akin to XML, and what my
software reads as akin to an XML DTD, if you're into that sort of
structured data format lingo.

The Stockholm markup tags that are parsed semantically by my software
are as follows:

\subsubsection{Recognized #=GF annotations}
\begin{wideitem}
\item [\emprog{ID  <s>}] 
	Identifier. \emprog{<s>} is a name for the alignment;
	e.g. ``rrm''. One word. Unique in file.

\item [\emprog{AC  <s>}]
	Accession. \emprog{<s>} is a unique accession number for the
	alignment; e.g. 
	``PF00001''. Used by the Pfam database, for instance. 
	Often a alphabetical prefix indicating the database
	(e.g. ``PF'') followed by a unique numerical accession.
	One word. Unique in file. 
	
\item [\emprog{DE  <s>}]
	Description. \emprog{<s>} is a free format line giving
	a description of the alignment; e.g.
	``RNA recognition motif proteins''. One line. Unique in file.

\item [\emprog{AU  <s>}]
	Author. \emprog{<s>} is a free format line listing the 
	authors responsible for an alignment; e.g. 
	``Bateman A''. One line. Unique in file.

\item [\emprog{GA  <f> <f>}]
	Gathering thresholds. Two real numbers giving HMMER bit score
	per-sequence and per-domain cutoffs used in gathering the
	members of Pfam full alignments. See Pfam and HMMER
	documentation for more detail.
	
\item [\emprog{NC  <f> <f>}]
	Noise cutoffs. Two real numbers giving HMMER bit score
	per-sequence and per-domain cutoffs, set according to the
	highest scores seen for unrelated sequences when gathering
	members of Pfam full alignments. See Pfam and HMMER
	documentation for more detail.

\item [\emprog{TC  <f> <f>}]
	Trusted cutoffs. Two real numbers giving HMMER bit score
	per-sequence and per-domain cutoffs, set according to the
	lowest scores seen for true homologous sequences that
	were above the GA gathering thresholds, when gathering
	members of Pfam full alignments. See Pfam and HMMER
	documentation for more detail.
\end{wideitem}

\subsection{Recognized #=GS annotations}

\begin{wideitem}
\item [\emprog{WT  <f>}]
	Weight. \emprog{<f>} is a positive real number giving the
	relative weight for a sequence, usually used to compensate
	for biased representation by downweighting similar sequences.	
	Usually the weights average 1.0 (e.g. the weights sum to
	the number of sequences in the alignment) but this is not
	required. Either every sequence must have a weight annotated, 
	or none	of them can.  

\item [\emprog{AC  <s>}]
	Accession. \emprog{<s>} is a database accession number for 
	this sequence. (Compare the \prog{#=GF AC} markup, which gives
	an accession for the whole alignment.) One word. 
	
\item [\emprog{DE  <s>}]
	Description. \emprog{<s>} is one line giving a description for
	this sequence. (Compare the \prog{#=GF DE} markup, which gives
	a description for the whole alignment.)
\end{wideitem}


\subsection{Recognized #=GC annotations}

\begin{wideitem}
\item [\emprog{RF}]
	Reference line. Any character is accepted as a markup for a
	column. The intent is to allow labeling the columns with some
	sort of mark.
	
\item [\emprog{SS_cons}]
	Secondary structure consensus. For protein alignments,
	DSSP codes or gaps are accepted as markup: [HGIEBTSCX.-_], where
	H is alpha helix, G is 3/10-helix, I is p-helix, E is extended
	strand, B is a residue in an isolated b-bridge, T is a turn, 
	S is a bend, C is a random coil or loop, and X is unknown
	(for instance, a residue that was not resolved in a crystal
	structure). For RNA alignments
	the symbols \verb+>+ and \verb+<+ are
	used for base pairs (pairs point at each other).  \verb-+- indicate
	definitely single-stranded positions, and any gap symbol indicates
	unassigned bases or single-stranded positions.  This description
	roughly follows \cite{Konings89}. 
	RNA pseudoknots are represented by alphabetic characters, with upper
	case letters representing the 5' side of the helix and lower case
	letters representing the 3' side. Note that this limits the
	annotation to a maximum of 26 pseudoknots per sequence.
	

\item [\emprog{SA_cons}]
	Surface accessibility consensus. 0-9, gap symbols, or X are
	accepted as markup. 0 means <10\% accessible residue surface
	area, 1 means <20\%, 9 means <100\%, etc. X means unknown
	structure.
\end{wideitem}

\subsection{Recognized #=GR annotations}

\begin{wideitem}
\item [\emprog{SS}]
	Secondary structure consensus. See \prog{#=GC SS_cons} above.
\item [\emprog{SA}]
	Surface accessibility consensus. See \prog{#=GC SA_cons} above.
\end{wideitem}