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#ifndef CLOSURES_H |
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#define CLOSURES_H |
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|
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/* |
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* closures.h |
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* TopHat |
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* |
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* Created by Cole Trapnell on 1/14/09. |
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* Copyright 2009 Cole Trapnell. All rights reserved. |
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* |
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*/ |
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|
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#ifdef HAVE_CONFIG_H |
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#include <config.h> |
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#endif |
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#include <set> |
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|
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#include "common.h" |
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#include "junctions.h" |
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|
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struct SpliceMotif |
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{ |
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SpliceMotif(uint32_t p, bool l) : pos(p), left_motif(l) {} |
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int pos : 31; |
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bool left_motif : 1; |
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|
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bool operator<(const SpliceMotif& rhs) const |
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{ |
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return this->pos < rhs.pos; |
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} |
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|
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bool operator<=(const SpliceMotif& rhs) const |
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{ |
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return this->pos <= rhs.pos; |
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} |
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}; |
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|
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// This simply finds all donor and acceptors in a given string, |
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// regardless of whether they are within or near an initially mapped |
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// region. |
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template<typename TStr> |
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class SimpleIntronFinder |
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{ |
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vector<SpliceMotif> _hits; |
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|
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public: |
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// This should take three TStr, and we should be using some search function |
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// of seqan to look up the hits. Don't ask. |
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SimpleIntronFinder(TStr& ref_str, const string& left_motif, const string& right_motif) |
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{ |
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_hits.clear(); |
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|
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for (size_t i = 0; i < length(ref_str) - 1; ++i) |
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{ |
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// Look at a slice of the reference without creating a copy. |
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seqan::Infix<RefSequenceTable::Sequence>::Type curr = seqan::infix(ref_str,i, i + 2); |
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if (curr == left_motif) |
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_hits.push_back(SpliceMotif(i, true)); |
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else if (curr == right_motif) |
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_hits.push_back(SpliceMotif(i, false)); |
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} |
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} |
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|
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const vector<size_t>& hits() const { return _hits; } |
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}; |
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|
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uint32_t searched = 0; |
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uint32_t closed = 0; |
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|
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typedef std::set<Junction, skip_count_lt > ClosureJunctionSet; |
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|
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template<typename TStr, typename IntronFinder> |
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class JunctionFinder |
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{ |
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public: |
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JunctionFinder(const IntronFinder& intron_finder, |
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uint32_t inner_dist_mean, |
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uint32_t inner_dist_std_dev, |
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uint32_t min_intron_length, |
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uint32_t min_exon_length, |
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uint32_t bowtie_overlap_padding = 0, |
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uint32_t max_gap = 0, |
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uint32_t max_juncs = 7500000 /*this is the max juncs from a single strand*/) : |
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_inner_dist_mean(inner_dist_mean), |
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_inner_dist_std_dev(inner_dist_std_dev), |
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_min_intron_len(min_intron_length), |
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_min_exon_len(min_exon_length), |
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_motif_hits(intron_finder.hits()), |
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_padding(bowtie_overlap_padding), |
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_max_gap(max_gap), |
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_max_juncs(max_juncs) |
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{ |
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_left_memo.resize(_motif_hits.size()); |
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_right_memo.resize(_motif_hits.size()); |
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} |
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|
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void possible_junctions(ClosureJunctionSet& potential_splices, |
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const BowtieHit& h1, |
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const BowtieHit& h2) |
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{ |
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assert (h1.ref_id() == h2.ref_id()); |
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|
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int minor_hit_start, major_hit_start; |
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int minor_hit_end, major_hit_end; |
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if (h1.left() < h2.left()) |
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{ |
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minor_hit_start = (int)h1.left(); |
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minor_hit_end = (int)h1.right(); |
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major_hit_start = (int)h2.left(); |
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major_hit_end = (int)h2.right(); |
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} |
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else |
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{ |
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minor_hit_start = (int)h2.left(); |
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minor_hit_end = (int)h2.right(); |
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major_hit_start = (int)h1.left(); |
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major_hit_end = (int)h1.right(); |
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} |
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|
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int inner_dist = major_hit_start - minor_hit_end; |
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|
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if (inner_dist > (int)max_closure_intron_length || |
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inner_dist < (int)min_closure_intron_length) |
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return; |
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// Bowtie allows some mismatches, so we need to allow for the fact that |
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// the donor/acceptor is actually "inside" the alignment for one or both |
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// ends of the insert. That is, intron may be longer than the pair's |
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// inner distance in the genomic coordinate space. This variable |
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// padding number accounts for this fact in a silly way that could be |
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// improved. |
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|
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int expected_inner_dist = _inner_dist_mean; |
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|
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if (abs(inner_dist - expected_inner_dist) <= (int)_inner_dist_std_dev) |
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return; |
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|
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++searched; |
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|
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if (search(potential_splices, h1.ref_id(), minor_hit_end, major_hit_start, expected_inner_dist)) |
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++closed; |
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} |
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|
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private: |
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|
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vector<SpliceMotif>::const_iterator _motif_upper_bound; |
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vector<SpliceMotif>::const_iterator _motif_lower_bound; |
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|
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/* The following computation identifies possible splice sites. |
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|
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|||||||||||||||-----------------------------------------|||||||||||||||| |
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read_len inner_dist read_len |
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Where r is the length of a read and x is the *internal* distance between |
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mates in the *genomic* coordinate space. We first check to see whether |
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x is too long to be an insert from a single contiguous substring of the |
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genome. That is, if x is longer than you'd expect from a unspliced |
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molecule, than the insert likely spans a splice junction. |
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|
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The expected value of x when the insert doesn't span a splice is the |
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library insert size mean (I) minus twice the read length, plus or minus |
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the library insert size standard deviation. If x is longer than that, |
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the insert probably spans a splice. |
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|
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For an insert that spans a splice junction, that junction must fall between |
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the ends of the mates. Let the right side of the alignment of the left |
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read be called minor_hit_end, and the left side of the alignment of the right |
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read be major_hit_start. Then an insert's inner_dist = major_hit_start - |
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minor_hit_end. Let the expected inner distance for a insert that doesn't |
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cross a junction be Insert_mean - 2* read_len. Then a splice-crossing insert |
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must have inner_dist >= expected_inner_dist. |
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|
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Let the actual splice position = (splice_left, splice_right). Then |
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(splice_left - minor_hit_end) + (major_hit_start - splice_right) = |
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expected_inner_dist +/- std_dev. |
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*/ |
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|
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/* |
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|
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The search for possible junctions works as follows. Given two genomic |
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coordinates, start, and ref_target, and an expected inner distance between |
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their projections in the transcriptome coordinate space, we can enumerate |
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paths through the genome that represent possible spliced transcript |
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segments and connect start and ref_target. That is, assuming start and |
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ref_target are both in exonic regions, we can find all possible |
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genomic subsequences formed by concatenating segments flanked by an "AG" on |
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the left and a "GT" on the right. The resulting concatentation is a |
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possible 'closure' if its length is approximately equal to the expected |
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inner distance between mate pairs for this library. |
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|
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To actually perform the search, this class uses two recursive functions |
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that call each other in an alternating fashion. search_hop_left_motif takes |
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a genomic coordinate lstart and finds paths to ref_target that start with |
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a "GT" that occurs in (lstart, ref_target]. search_hop_right_motif takes a |
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genomic coordinate rstart and finds paths to ref_target that start with an |
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"AG" that occurs in (rstart, ref_target]. A valid path from start to |
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ref_target must have a "GT" on the left and an "AG" on the right, so valid |
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paths may only be found in a call to search_hop_right_motif. |
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|
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*/ |
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|
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bool search(ClosureJunctionSet& splices, |
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uint32_t ref_id, |
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uint32_t start, |
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uint32_t ref_target, |
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int expected_inner_dist) |
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{ |
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uint32_t left_start_pos = max((int)start - (int)_padding, 0); |
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uint32_t right_end_pos = ref_target + _padding; |
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|
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SpliceMotif left_start(left_start_pos, true); |
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SpliceMotif right_end(right_end_pos, false); |
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|
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|
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_motif_lower_bound = lower_bound(_motif_hits.begin(), |
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_motif_hits.end(), |
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left_start); |
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|
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_motif_upper_bound = upper_bound(_motif_hits.begin(), |
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_motif_hits.end(), |
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right_end); |
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|
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//fprintf(stderr, "Curr junctions = %d\n", splices.size()); |
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|
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/* TODO: consider some kind of long-term memo. Closures found at the |
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top level of recursion here can be saved and potentially reused in |
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future searches on overlapping intervals. |
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*/ |
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bool c = false; |
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if (search_hop_left_motif(splices, |
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ref_id, |
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_motif_lower_bound, |
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right_end_pos, |
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expected_inner_dist, |
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0, |
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true) != -1) |
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c = true; |
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|
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/* Clear left short-term memo */ |
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vector<vector<int> >::iterator _lm_begin = _left_memo.begin() + |
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(_motif_lower_bound - _motif_hits.begin()); |
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|
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vector<vector<int> >::iterator _lm_end = _left_memo.begin() + |
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(_motif_upper_bound - _motif_hits.begin()) + 1; |
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|
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_lm_end = min(_lm_end, _left_memo.end()); |
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|
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/* Clear right short-term memo */ |
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vector<vector<int> >::iterator _rm_begin = _right_memo.begin() + |
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(_motif_lower_bound - _motif_hits.begin()); |
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|
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vector<vector<int> >::iterator _rm_end = _right_memo.begin() + |
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(_motif_upper_bound - _motif_hits.begin()) + 1; |
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|
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_rm_end = min(_rm_end, _right_memo.end()); |
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|
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if (_lm_begin < _lm_end) |
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fill(_lm_begin, _lm_end, vector<int>()); |
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|
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if (_rm_begin < _rm_end) |
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fill(_rm_begin, _rm_end, vector<int>()); |
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|
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while(splices.size() > _max_juncs) |
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{ |
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splices.erase(*splices.rbegin()); |
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} |
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|
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return c; |
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} |
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|
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inline int _abs(int x) |
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{ |
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return (x > 0) ? x : -x; |
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} |
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|
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inline int check_memo(const vector<vector<int> >& memo, |
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size_t motif_pos, |
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int curr_path_distance, |
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int target_path_distance, |
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int tolerance) |
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{ |
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const vector<int>& distances = memo[motif_pos]; |
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for (size_t i = 0; i < distances.size(); ++i) |
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{ |
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int dist = distances[i]; |
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if (_abs(curr_path_distance + dist - target_path_distance) < tolerance) |
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return dist; |
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} |
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return -1; |
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} |
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|
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int search_hop_left_motif(ClosureJunctionSet& splices, |
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uint32_t ref_id, |
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vector<SpliceMotif>::const_iterator start, |
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int ref_target, |
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int expected_inner_dist, |
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uint32_t curr_path_length, |
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bool initial_hop = false) |
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{ |
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int in_valid_path = -1; |
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// SpliceMotif left_bound(start + (initial_hop ? 0 : _min_exon_len), true); |
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// SpliceMotif right_bound(start + (expected_inner_dist - curr_path_length) + (2 * _padding) + _max_gap, true); |
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|
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vector<SpliceMotif>::const_iterator lb_left = start; |
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|
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while (lb_left <= _motif_upper_bound && |
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lb_left < _motif_hits.end() && |
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(lb_left->pos - start->pos < (initial_hop ? 0 :(int) _min_exon_len) || !lb_left->left_motif)) |
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{ |
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lb_left++; |
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} |
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|
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|
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for (vector<SpliceMotif>::const_iterator li = lb_left; |
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li != _motif_hits.end(); |
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++li) |
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{ |
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if (!li->left_motif) //skip over the right motifs |
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continue; |
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//assert (*li >= SpliceMotif(start, true) || initial_hop); |
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int dist = (int)li->pos - (int)start->pos; |
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int next_path_len = curr_path_length + dist; |
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|
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int tolerance = _inner_dist_std_dev + 2 * _padding + _max_gap; |
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|
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if (next_path_len - expected_inner_dist > tolerance) |
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{ |
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return in_valid_path; |
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} |
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|
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int next = li->pos; |
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//assert(next + _min_intron_len <= ref_target); |
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if(next + (int)_min_intron_len <= ref_target) |
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{ |
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int ret = -1; |
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ret = check_memo(_left_memo, |
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li - _motif_hits.begin(), |
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curr_path_length + dist, |
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expected_inner_dist, |
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tolerance); |
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if (ret != -1) |
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{ |
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in_valid_path = ret + dist; |
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_left_memo[li - _motif_hits.begin()].push_back(in_valid_path); |
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} |
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else |
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{ |
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ret = search_hop_right_motif(splices, |
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ref_id, |
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li, |
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ref_target, |
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expected_inner_dist, |
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next_path_len); |
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if (ret != -1) |
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{ |
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in_valid_path = ret + dist; |
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_left_memo[li - _motif_hits.begin()].push_back(in_valid_path); |
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} |
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} |
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} |
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|
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} |
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|
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return in_valid_path; |
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|
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} |
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|
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int search_hop_right_motif(ClosureJunctionSet& splices, |
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uint32_t ref_id, |
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vector<SpliceMotif>::const_iterator start, |
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int ref_target, |
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int expected_inner_dist, |
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uint32_t curr_path_length) |
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{ |
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int in_valid_path = -1; |
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|
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// SpliceMotif left_start(start + _min_intron_len - 2, false); |
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// SpliceMotif right_start(ref_target, false); |
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if (ref_target < start->pos) |
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{ |
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return in_valid_path; |
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} |
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vector<SpliceMotif>::const_iterator lb_right = start; |
| 382 |
while (lb_right <= _motif_upper_bound && |
| 383 |
lb_right < _motif_hits.end() && |
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(lb_right->pos - start->pos < (int)_min_intron_len - 2 || lb_right->left_motif)) |
| 385 |
{ |
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lb_right++; |
| 387 |
} |
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|
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for (vector<SpliceMotif>::const_iterator ri = lb_right; |
| 390 |
ri != _motif_hits.end(); |
| 391 |
++ri) |
| 392 |
{ |
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if (ri->left_motif) //skip over the left motifs |
| 394 |
continue; |
| 395 |
if (ri->pos > ref_target) |
| 396 |
{ |
| 397 |
return in_valid_path; |
| 398 |
} |
| 399 |
int next = ri->pos + 2; |
| 400 |
int final_hop_dist = (int)ref_target - (int)next; |
| 401 |
int final_path_len = curr_path_length + final_hop_dist; |
| 402 |
|
| 403 |
int tolerance = _inner_dist_std_dev + 2 * _padding + _max_gap; |
| 404 |
|
| 405 |
if (abs(expected_inner_dist - final_path_len) <= tolerance) |
| 406 |
{ |
| 407 |
splices.insert(Junction(ref_id, |
| 408 |
start->pos - 1, |
| 409 |
next, |
| 410 |
false, |
| 411 |
start->pos - 1 - next)); |
| 412 |
in_valid_path = final_hop_dist; |
| 413 |
_right_memo[ri - _motif_hits.begin()].push_back(final_hop_dist); |
| 414 |
} |
| 415 |
else |
| 416 |
{ |
| 417 |
int ret = -1; |
| 418 |
ret = check_memo(_right_memo, |
| 419 |
ri - _motif_hits.begin(), |
| 420 |
curr_path_length, |
| 421 |
expected_inner_dist, |
| 422 |
tolerance); |
| 423 |
if (ret != -1) |
| 424 |
{ |
| 425 |
splices.insert(Junction(ref_id, |
| 426 |
start->pos - 1, |
| 427 |
next, |
| 428 |
false, |
| 429 |
start->pos - 1 - next)); |
| 430 |
|
| 431 |
in_valid_path = ret; |
| 432 |
_right_memo[ri - _motif_hits.begin()].push_back(ret); |
| 433 |
} |
| 434 |
else |
| 435 |
{ |
| 436 |
|
| 437 |
ret = search_hop_left_motif(splices, |
| 438 |
ref_id, |
| 439 |
ri, |
| 440 |
ref_target, |
| 441 |
expected_inner_dist, |
| 442 |
curr_path_length); |
| 443 |
if (ret != -1) |
| 444 |
{ |
| 445 |
splices.insert(Junction(ref_id, |
| 446 |
start->pos - 1, |
| 447 |
next, |
| 448 |
false, |
| 449 |
start->pos - 1 - next)); |
| 450 |
|
| 451 |
in_valid_path = ret; |
| 452 |
_right_memo[ri - _motif_hits.begin()].push_back(in_valid_path); |
| 453 |
} |
| 454 |
} |
| 455 |
} |
| 456 |
} |
| 457 |
|
| 458 |
return in_valid_path; |
| 459 |
} |
| 460 |
|
| 461 |
uint32_t _inner_dist_mean; |
| 462 |
uint32_t _inner_dist_std_dev; |
| 463 |
uint32_t _min_intron_len; |
| 464 |
uint32_t _min_exon_len; |
| 465 |
|
| 466 |
vector<SpliceMotif> _motif_hits; |
| 467 |
uint32_t _padding; |
| 468 |
uint32_t _max_gap; |
| 469 |
uint32_t _max_juncs; |
| 470 |
vector<vector<int> > _left_memo; |
| 471 |
vector<vector<int> > _right_memo; |
| 472 |
}; |
| 473 |
|
| 474 |
typedef std::set<pair<size_t, size_t> > CoordSet; |
| 475 |
void check_mates(const HitList& hits1_in_ref, |
| 476 |
const HitList& hits2_in_ref, |
| 477 |
vector<pair<size_t, size_t> >& happy_mates, |
| 478 |
vector<size_t>& map1_singletons, |
| 479 |
vector<size_t>& map2_singletons); |
| 480 |
|
| 481 |
#endif |
