/**********************************************************************
shuffle_sequence()
shuffle a given sequences based on their content
**********************************************************************/
void shuffle_sequence(
SEQ_T* seq, /* original sequence IN */
unsigned int seed, /* seed IN */
SEQ_T** target /* target sequence OUT */
){
my_srand(seed);
assert(*target==NULL);
// reset target if not null
if (*target != NULL){
free_seq(*target);
}
*target = allocate_seq(get_seq_name(seq),"shuffled",get_seq_offset(seq),get_raw_sequence(seq));
char *raw = get_raw_sequence(*target);
/* copy original in temp string */
char* tmp = (char*)mm_calloc(get_seq_length(seq)+1,sizeof(char));
strcpy(tmp,get_raw_sequence(seq));
tmp[get_seq_length(seq)]='\0';
int i,j;
char *ss;
char *dd;
for(j=0,i=get_seq_length(seq);i>0;i--){
// Pick a random number in the range:
int pick = rand() % i;
raw[j++] = tmp[pick];
// "shift" routine here eliminates the "picked" base from the _src string:
// dd starts at the picked position: ss is one beyond that:
for( dd = tmp+pick , ss = dd + 1 ; *dd ; *dd++=*ss++ );
}
myfree(tmp);
}
/**
* Creates a motif for a given mod using a simple frequency matrix.
*/
void create_simple_motif(SUMMARY_T* summary,
MOMO_OPTIONS_T* options,
MOD_INFO_T * mod_info) {
int i;
int j;
const char* alph_letters = summary->alph_letters;
// Create the frequency matrix
MATRIX_T* freqs = NULL;
freqs = get_count_matrix(freqs, mod_info->seq_list, NULL, options, summary);
normalize_rows(0.0, freqs);
// Create the motif
MOTIF_INFO_T* motifinfo = mm_malloc(sizeof(MOTIF_INFO_T));
motifinfo->motif = allocate_motif(mod_info->mod_name, "", summary->alph, freqs, NULL);
motifinfo->seqs = arraylst_create();
for (i = 0; i < arraylst_size(mod_info->seq_list); ++i) {
SEQ_T* seqobject = options->eliminate_repeats ? hash_get_entry_value(arraylst_get(i, mod_info->seq_list)) : arraylst_get(i, mod_info->seq_list);
arraylst_add(get_raw_sequence(seqobject), motifinfo->seqs);
}
motifinfo->fg_size = arraylst_size(mod_info->seq_list);
arraylst_add(motifinfo, mod_info->motifinfos);
// clean up
free_matrix(freqs);
}
/****************************************************************************
* Create a lookup table for converting an index into a sequence to an index
* into the alignment. Note that because there are many alignment positions
* that correspond to a sequence position we take the first occurence.
* JCH: I have added this function for the sake of the BLS scan mode
* so that single mode matches in each sequence can be mapped back
* to positions in the alignment.
****************************************************************************/
int* make_seq_to_alignment_table(int ref_seq_index, ALIGNMENT_T* an_alignment) {
char* raw_seq = NULL;
int align_length = 0;
int align_index = 0;
int seq_index = 0;
int *table = NULL;
SEQ_T* seq = NULL;
align_length = get_alignment_length(an_alignment);
seq = get_alignment_sequence(ref_seq_index, an_alignment);
raw_seq = get_raw_sequence(seq);
// Table is indexed by position in the sequence
// Table values are the first corresponding
// position in the alignment.
table = (int *) mm_malloc((align_length) * sizeof(int));
seq_index = 0;
table[seq_index] = 0;
for (align_index = 0; align_index < align_length; align_index++) {
if (raw_seq[align_index] != '-' && raw_seq[align_index] != '.') {
seq_index++;
table[seq_index] = align_index;
}
}
return table;
}
void print_phylip_alignment
(ALIGNMENT_T* the_alignment,
FILE* outfile)
{
int i_seq;
int i_position;
char buffer[OUTPUT_WIDTH+1];
char* this_sequence;
fprintf(outfile, "%d %d\n", the_alignment->num_sequences,
the_alignment->length);
/* Print the IDs and initial sequences. */
for (i_seq = 0; i_seq < the_alignment->num_sequences; i_seq++) {
/* Print the ID. */
strncpy(buffer, get_seq_name(the_alignment->sequences[i_seq]), 10);
buffer[10] = '\0';
fprintf(outfile, "%-10s ", buffer);
/* Print the first block of sequence. */
this_sequence = get_raw_sequence(the_alignment->sequences[i_seq]);
strncpy(buffer, &(this_sequence[0]), OUTPUT_WIDTH);
buffer[OUTPUT_WIDTH] = '\0';
fprintf(outfile, "%s\n", buffer);
}
/* Blank line between sequences. */
fprintf(outfile, "\n");
/* Print successive blocks. */
for (i_position = OUTPUT_WIDTH; i_position < the_alignment->length;
i_position += OUTPUT_WIDTH) {
for (i_seq = 0; i_seq < the_alignment->num_sequences; i_seq++) {
this_sequence = get_raw_sequence(the_alignment->sequences[i_seq]);
strncpy(buffer, &(this_sequence[i_position]), OUTPUT_WIDTH);
buffer[OUTPUT_WIDTH] = '\0';
fprintf(outfile, " %s\n", buffer);
}
/* Blank line between sequences. */
fprintf(outfile, "\n");
}
}
char* get_raw_subsequence
(int start, int stop, SEQ_T* a_sequence)
{
assert(a_sequence != NULL);
assert((stop - start) >= 0);
char *sequence = get_raw_sequence(a_sequence);
char *subsequence = mm_malloc((stop - start + 2) * sizeof(char));
strncpy(subsequence, sequence + start, stop - start + 1);
subsequence[stop - start + 1] = 0;
return(subsequence);
}
/****************************************************************************
* Extract a small alignment out of the middle of a larger alignment.
****************************************************************************/
ALIGNMENT_T* extract_subalignment
(int start,
int width,
ALIGNMENT_T* alignment)
{
int num_sequences = get_num_aligned_sequences(alignment);
SEQ_T** sequences = get_alignment_sequences(alignment);
SEQ_T** subsequences = (SEQ_T**)mm_malloc(num_sequences * sizeof(SEQ_T*));
// Extract the specified columns into a new list of sequences.
int i_seq = 0;
char* subsequence = mm_malloc((width + 1) * sizeof(char));
for (i_seq = 0; i_seq < num_sequences; i_seq++) {
SEQ_T* this_seq = sequences[i_seq];
char* raw_seq = get_raw_sequence(this_seq);
strncpy(subsequence, raw_seq + start, width);
subsequence[width] = '\0';
subsequences[i_seq] =
allocate_seq(get_seq_name(this_seq),
get_seq_description(this_seq),
get_seq_offset(this_seq),
subsequence);
}
// Extract the consensus string in the specified columns.
char* consensus = get_consensus_string(alignment);
char* subconsensus = mm_malloc(sizeof(char) * (width + 1));
strncpy(subconsensus, consensus + start, width);
subconsensus[width] = '\0';
// Allocate and return the new alignment.
ALIGNMENT_T* subalignment
= allocate_alignment(get_alignment_name(alignment),
get_alignment_description(alignment),
num_sequences,
subsequences,
subconsensus);
// Free local dynamic memory.
for (i_seq = 0; i_seq < num_sequences; i_seq++) {
free_seq(subsequences[i_seq]);
}
myfree(subsequences);
myfree(subsequence);
return(subalignment);
}
static double * log_cumulative_background(ALPH_T *alph, const int sdbg_order, SEQ_T *sequence) {
BGCALC_T *calc;
ARRAY_T *cp;
double *logcumback;
const char *raw_seq;
int i;
if (sdbg_order < 0) die("No such thing as a negative background order");
logcumback = mm_malloc(sizeof(double) * (get_seq_length(sequence)+1));
raw_seq = get_raw_sequence(sequence);
calc = NULL;
// calculate background model
calculate_markov_model(alph, sdbg_order, 1.0, false, raw_seq, &calc);
cp = calculate_markov_model(alph, sdbg_order, 1.0, false, NULL, &calc);
// add x-tuples to model
extend_markov_model(alph, true, SUM_FREQS, cp);
// normalize for each prefix (convert to conditional probability)
for (i = 0; i < get_array_length(cp); i += alph_size_wild(alph)) {
normalize_subarray(i, alph_size_core(alph), 0, cp);
set_array_item(i + alph_wild(alph), 1.0, cp);
}
calculate_log_cumulative_background(alph, true, sdbg_order, cp, raw_seq, logcumback);
free_array(cp);
return logcumback;
}
/**
* Remove sequences do not match a pattern from phospho and bg lists and update their respective count matrix
*/
void remove_sequences_and_update_matrix(char letter,
int pos,
ARRAYLST_T* seqs,
MOTIFX_STATUS_T** status_array,
int* num_active,
MATRIX_T* count,
SUMMARY_T* summary,
MOMO_OPTIONS_T* options) {
int i;
const char* alph_letters = summary->alph_letters;
// Look through phospho_seqs and remove sequences. Update phospho_seqs
for (i = 0; i < arraylst_size(seqs); ++i) {
char* curr_seq = get_raw_sequence((SEQ_T*) (options->eliminate_repeats ? hash_get_entry_value((HASH_TABLE_ENTRY*) arraylst_get(i, seqs)) : arraylst_get(i, seqs)));
// For anything active that does not match the pattern, turn it inactive.
MOTIFX_STATUS_T status = (*status_array)[i];
if (status == ACTIVE && curr_seq[pos] != letter) {
*num_active = *num_active - 1;
(*status_array)[i] = INACTIVE;
}
}
count = get_count_matrix(count, seqs, status_array, options, summary);
}
/*************************************************************************
* Calculate the odds score for each motif-sized window at each
* site in the sequence using the given nucleotide frequencies.
*
* This function is a lightweight version based on the one contained in
* motiph-scoring. Several calculations that are unnecessary for gomo
* have been removed in order to speed up the process
*************************************************************************/
static double score_sequence(
SEQ_T *seq, // sequence to scan (IN)
MOTIF_T *motif, // motif already converted to odds values (IN)
PSSM_T *m_pssm, // motif pssm (IN)
MATRIX_T *m_odds, // motif odds (IN)
int method, // method used for scoring (IN)
double threshold, // Threshold to use in TOTAL_HITS mode with a PWM
ARRAY_T *bg_freqs //background model
)
{
assert(seq != NULL);
assert(motif != NULL);
assert((method == TOTAL_HITS && m_pssm) || (method != TOTAL_HITS && m_odds));
char* raw_seq = get_raw_sequence(seq);
int seq_length = get_seq_length(seq);
// Get the pv lookup table
ARRAY_T* pv_lookup = NULL;
if (NULL != m_pssm) {
pv_lookup = m_pssm->pv;
assert(get_array_length(pv_lookup) > 0);
}
// Prepare storage for the string representing the portion
// of the reference sequence within the window.
char* window_seq = (char *) mm_malloc(sizeof(char) * (get_motif_length(motif) + 1));
window_seq[get_motif_length(motif)] = '\0';
int max_index = seq_length - get_motif_length(motif);
if (max_index < 0) max_index = 0;
const int asize = alph_size(get_motif_alph(motif), ALPH_SIZE);
double* odds = (double*) mm_malloc(sizeof(double)*max_index);
double* scaled_log_odds = (double*) mm_malloc(sizeof(double)*max_index);
// For each site in the sequence
int seq_index;
for (seq_index = 0; seq_index < max_index; seq_index++) {
double odd = 1.0;
scaled_log_odds[seq_index] = 0;
// For each site in the motif window
int motif_position;
for (motif_position = 0; motif_position < get_motif_length(motif); motif_position++) {
char c = raw_seq[seq_index + motif_position];
window_seq[motif_position] = c;
// Check for gaps at this site
if(c == '-' || c == '.') {
break;
}
// Check for ambiguity codes at this site
//TODO: This next call is very expensive - it takes up approx. 10% of a
// programme's running time. It should be fixed up somehow.
int aindex = alph_index(get_motif_alph(motif), c);
if (aindex > asize) {
break;
}
if (method == TOTAL_HITS) {
//If we're in this mode, then we're using LOG ODDS.
//scaled_log_odds[seq_index] += get_matrix_cell(motif_position, aindex, get_motif_freqs(motif));
scaled_log_odds[seq_index] += get_matrix_cell(motif_position, aindex, m_pssm->matrix);
} else {
odd *= get_matrix_cell(motif_position, aindex, m_odds);
}
}
odds[seq_index] = odd;
}
// return odds as requested (MAX or AVG scoring)
double requested_odds = 0.0;
if (method == AVG_ODDS){
for (seq_index = 0; seq_index < max_index; seq_index++) {
requested_odds += odds[seq_index];
}
requested_odds /= max_index + 1; // Divide by 0 if max_index==0
} else if (method == MAX_ODDS){
for (seq_index = 0; seq_index < max_index; seq_index++) {
if (odds[seq_index] > requested_odds){
requested_odds = odds[seq_index];
}
}
} else if (method == SUM_ODDS) {
for (seq_index = 0; seq_index < max_index; seq_index++) {
requested_odds += odds[seq_index];
}
} else if (method == TOTAL_HITS) {
for (seq_index = 0; seq_index < max_index; seq_index++) {
if (scaled_log_odds[seq_index] >= (double)get_array_length(pv_lookup)) {
scaled_log_odds[seq_index] = (double)(get_array_length(pv_lookup) - 1);
}
double pvalue = get_array_item((int) scaled_log_odds[seq_index], pv_lookup);
//Figure out how to calculate the p-value of a hit
//fprintf(stderr, "m: %s pv_l len: %i scaled_log_odds: %g seq index: %i pvalue: %g\n",
// get_motif_id(motif), get_array_length(pv_lookup), scaled_log_odds[seq_index], seq_index, pvalue);
if (pvalue < threshold) {
requested_odds++; //Add another hit.
}
if (verbosity > HIGHER_VERBOSE) {
fprintf(stderr, "Window Data: %s\t%s\t%i\t%g\t%g\t%g\n",
get_seq_name(seq), get_motif_id(motif), seq_index, scaled_log_odds[seq_index], pvalue, threshold);
}
}
}
myfree(odds);
myfree(scaled_log_odds);
myfree(window_seq);
return requested_odds;
}
/**
* Recursive function. Creates and stores a motif using the motif-x
* algorithm until no more are left.
*/
void create_motifx_motif(ARRAYLST_T* phospho_seqs,
ARRAYLST_T* bg_seqs,
MOTIFX_STATUS_T** phospho_status,
MOTIFX_STATUS_T** bg_status,
MATRIX_T* phospho_count,
MATRIX_T* bg_count,
int* num_active,
int* num_bg_active,
char* modname,
MOD_INFO_T* mod_info,
MOMO_OPTIONS_T* options,
SUMMARY_T* summary) {
int i;
int j;
const char* alph_letters = summary->alph_letters;
// Initialize pattern, sequence count, bg sequence count, and overall score for this motif.
char* pattern = mm_malloc(options->width + 1);
for (i = 0; i < options->width; ++i) {
pattern[i] = 'X';
}
pattern[options->width] = '\0';
int* num_active_copy = mm_malloc(sizeof(int));
*num_active_copy = *num_active;
int* num_bg_active_copy = mm_malloc(sizeof(int));
*num_bg_active_copy = *num_bg_active;
double* motif_score = mm_malloc(sizeof(double));
*motif_score = 0;
// Set the pattern, num active copy, num bg active copy, motif score, and get a count of the sequences
MATRIX_T* result_count_matrix = add_to_pattern(pattern, phospho_seqs, bg_seqs, phospho_status, bg_status, num_active_copy, num_bg_active_copy, phospho_count, bg_count, motif_score, summary, options);
// If any of the characters are not X, then we have found a pattern
BOOLEAN_T found_pattern = FALSE;
for (i = 0; i < options->width; ++i) {
if (pattern[i] != 'X') {
found_pattern = TRUE;
}
}
// If there is a pattern, store the pattern and call create_motifx_motif again.
if (found_pattern) {
// fill out the rest of the pattern (e.g. if you have pattern ..ASAAA, and realize the actual pattern is A.ASAAA
for (i = 0; i < options->width; i++) {
for (j = 0; j < strlen(alph_letters); j++) {
if ((int) get_matrix_cell_defcheck(i, j, result_count_matrix) == *num_active_copy) {
pattern[i] = alph_letters[j];
}
}
}
// create the pattern name
char* pattern_name = mm_malloc(strlen(pattern) + strlen(modname) + 3);
pattern_name[0] = '\0';
strncat(pattern_name, pattern, strlen(pattern)/2);
strncat(pattern_name, "_", 1);
strncat(pattern_name, modname, strlen(modname));
strncat(pattern_name, "_", 1);
strncat(pattern_name, pattern + strlen(pattern)/2 + 1, strlen(pattern)/2);
// convert this count matrix into frequencies
normalize_rows(0.0, result_count_matrix);
// Store this motif
MOTIF_INFO_T* motifinfo = mm_malloc(sizeof(MOTIF_INFO_T));
MOTIF_T* motif = allocate_motif(pattern_name, "", summary->alph, result_count_matrix, NULL);
set_motif_nsites(motif, *num_active_copy);
motifinfo->motif = motif;
motifinfo->seqs = arraylst_create();
motifinfo->score = *motif_score;
motifinfo->fg_match = *num_active_copy;
motifinfo->fg_size = *num_active;
motifinfo->bg_match = *num_bg_active_copy;
motifinfo->bg_size = *num_bg_active;
for (i = 0; i < arraylst_size(phospho_seqs); ++i) {
MOTIFX_STATUS_T status = (*phospho_status)[i];
if (status == ACTIVE) {
SEQ_T* active_sequence = (options->eliminate_repeats) ? hash_get_entry_value(arraylst_get(i, phospho_seqs)) : arraylst_get(i, phospho_seqs);
arraylst_add(get_raw_sequence(active_sequence), motifinfo->seqs);
}
}
arraylst_add(motifinfo, mod_info->motifinfos);
// delete the sequences from this motif. turn inactive into active.
delete_sequences(phospho_status, arraylst_size(phospho_seqs));
delete_sequences(bg_status, arraylst_size(bg_seqs));
// update the count of number of actives
*num_active = *num_active - *num_active_copy;
*num_bg_active = *num_bg_active - *num_bg_active_copy;
// recalculate phospho count and bg count.
phospho_count = get_count_matrix(phospho_count, phospho_seqs, phospho_status, options, summary);
bg_count = get_count_matrix(bg_count, bg_seqs, bg_status, options, summary);
// free up space
myfree(pattern);
myfree(num_active_copy);
myfree(num_bg_active_copy);
myfree(motif_score);
myfree(pattern_name);
// try to create another motif.
create_motifx_motif(phospho_seqs,
bg_seqs,
phospho_status,
bg_status,
phospho_count,
bg_count,
num_active,
num_bg_active,
modname,
mod_info,
options,
summary);
}
// free up space
myfree(pattern);
myfree(num_active_copy);
myfree(num_bg_active_copy);
myfree(motif_score);
}
/*************************************************************************
* Entry point for ama
*************************************************************************/
int main(int argc, char *argv[]) {
int max_seq_length = MAX_SEQ;
STRING_LIST_T* selected_motifs = NULL;
double pseudocount = 0.01;
int output_format = CISML_FORMAT;
program_name = "ama";
int scoring = AVG_ODDS;
BOOLEAN_T pvalues = FALSE;
BOOLEAN_T normalize_scores = FALSE;
BOOLEAN_T combine_duplicates = FALSE;
int num_gc_bins = 1;
int sdbg_order = -1; // don't use sequence background
BOOLEAN_T scan_both_strands = TRUE;
ARRAY_T* pos_bg_freqs = NULL;
ARRAY_T* rev_bg_freqs = NULL;
clock_t c0, c1; /* measuring cpu_time */
CISML_T *cisml;
char * out_dir = NULL;
BOOLEAN_T clobber = FALSE;
int i;
int last = 0;
ALPH_T alph = INVALID_ALPH;
/**********************************************
* COMMAND LINE PROCESSING
**********************************************/
const int num_options = 16;
cmdoption const motif_scan_options[] = {
{ "max-seq-length", REQUIRED_VALUE },
{ "motif", REQUIRED_VALUE },
{ "motif-pseudo", REQUIRED_VALUE },
{ "rma", NO_VALUE },
{ "pvalues", NO_VALUE },
{ "sdbg", REQUIRED_VALUE },
{ "norc", NO_VALUE },
{ "cs", NO_VALUE },
{ "o-format", REQUIRED_VALUE },
{ "o", REQUIRED_VALUE },
{ "oc", REQUIRED_VALUE },
{ "scoring", REQUIRED_VALUE },
{ "verbosity", REQUIRED_VALUE },
{ "gcbins", REQUIRED_VALUE },
{ "last", REQUIRED_VALUE },
{ "version", NO_VALUE }
};
int option_index = 0;
// Define the usage message.
char usage[] = "USAGE: ama [options] <motif file> <sequence file> [<background file>]\n"
"\n"
" Options:\n"
" --sdbg <order>\t\t\tUse Markov background model of\n"
" \t\t\t\t\torder <order> derived from the sequence\n"
" \t\t\t\t\tto compute its likelihood ratios.\n"
" \t\t\t\t\tOverrides --pvalues, --gcbins and --rma;\n"
" \t\t\t\t\t<background file> is required unless\n"
" \t\t\t\t\t--sdbg is given.\n"
" --motif <id>\t\t\tUse only the motif identified by <id>.\n"
" \t\t\t\t\tThis option may be repeated.\n"
" --motif-pseudo <float>\t\tThe value <float> times the background\n"
" \t\t\t\t\tfrequency is added to the count of each\n"
" \t\t\t\t\tletter when creating the likelihood \n"
" \t\t\t\t\tratio matrix (default: %g).\n"
" --norc\t\t\t\tDisables the scanning of the reverse\n"
" \t\t\t\t\tcomplement strand.\n"
" --scoring [avg-odds|max-odds]\tIndicates whether the average or \n"
" \t\t\t\t\tthe maximum odds should be calculated\n"
" \t\t\t\t\t(default: avg-odds)\n"
" --rma\t\t\t\tScale motif scores to the range 0-1.\n"
" \t\t\t\t\t(Relative Motif Affinity).\n"
" \t\t\t\t\tMotif scores are scaled by the maximum\n"
" \t\t\t\t\tscore achievable by that PWM. (default:\n"
" \t\t\t\t\tmotif scores are not normalized)\n"
" --pvalues\t\t\t\tPrint p-value of avg-odds score in cisml\n"
" \t\t\t\t\toutput. Ignored for max-odds scoring.\n"
" \t\t\t\t\t(default: p-values are not printed)\n"
" --gcbins <bins>\t\t\tCompensate p-values for GC content of\n"
" \t\t\t\t\teach sequence using given number of \n"
" \t\t\t\t\tGC range bins. Recommended bins: 41.\n"
" \t\t\t\t\t(default: p-values are based on\n"
" \t\t\t\t\tfrequencies in background file)\n"
" --cs\t\t\t\tEnable combining sequences with same\n"
" \t\t\t\t\tidentifier by taking the average score\n"
" \t\t\t\t\tand the Sidac corrected p-value.\n"
" --o-format [gff|cisml]\t\tOutput file format (default: cisml)\n"
" \t\t\t\t\tignored if --o or --oc option used\n"
" --o <directory>\t\t\tOutput all available formats to\n"
" \t\t\t\t\t<directory>; give up if <directory>\n"
" \t\t\t\t\texists\n"
" --oc <directory>\t\t\tOutput all available formats to\n"
" \t\t\t\t\t<directory>; if <directory> exists\n"
" \t\t\t\t\toverwrite contents\n"
" --verbosity [1|2|3|4]\t\tControls amount of screen output\n"
" \t\t\t\t\t(default: %d)\n"
" --max-seq-length <int>\t\tSet the maximum length allowed for \n"
" \t\t\t\t\tinput sequences. (default: %d)\n"
" --last <int>\t\t\tUse only scores of (up to) last <n>\n"
" \t\t\t\t\tsequence positions to compute AMA.\n"
" --version \t\t\tPrint version and exit.\n"
"\n";
// Parse the command line.
if (simple_setopt(argc, argv, num_options, motif_scan_options) != NO_ERROR) {
die("Error processing command line options: option name too long.\n");
}
BOOLEAN_T setoutputformat = FALSE;
BOOLEAN_T setoutputdirectory = FALSE;
while (TRUE) {
int c = 0;
char* option_name = NULL;
char* option_value = NULL;
const char * message = NULL;
// Read the next option, and break if we're done.
c = simple_getopt(&option_name, &option_value, &option_index);
if (c == 0) {
break;
} else if (c < 0) {
(void) simple_getopterror(&message);
die("Error processing command line options (%s).\n", message);
} else if (strcmp(option_name, "max-seq-length") == 0) {
max_seq_length = atoi(option_value);
} else if (strcmp(option_name, "norc") == 0) {
scan_both_strands = FALSE;
} else if (strcmp(option_name, "cs") == 0) {
combine_duplicates = TRUE;
} else if (strcmp(option_name, "motif") == 0) {
if (selected_motifs == NULL) {
selected_motifs = new_string_list();
}
add_string(option_value, selected_motifs);
} else if (strcmp(option_name, "motif-pseudo") == 0) {
pseudocount = atof(option_value);
} else if (strcmp(option_name, "o-format") == 0) {
if (setoutputdirectory) {
if (verbosity >= NORMAL_VERBOSE)
fprintf(stderr, "output directory specified, ignoring --o-format\n");
} else {
setoutputformat = TRUE;
if (strcmp(option_value, "gff") == 0)
output_format = GFF_FORMAT;
else if (strcmp(option_value, "cisml") == 0)
output_format = CISML_FORMAT;
else {
if (verbosity >= NORMAL_VERBOSE)
fprintf(stderr, "Output format not known. Using standard instead (cisML).\n");
output_format = CISML_FORMAT;
}
}
} else if (strcmp(option_name, "o") == 0 || strcmp(option_name, "oc") == 0) {
setoutputdirectory = TRUE;
if (setoutputformat) {
if (verbosity >= NORMAL_VERBOSE)
fprintf(stderr, "output directory specified, ignoring --o-format\n");
}
clobber = strcmp(option_name, "oc") == 0;
out_dir = (char*) malloc (sizeof(char)*(strlen(option_value)+1));
strcpy(out_dir, option_value);
output_format = DIRECTORY_FORMAT;
} else if (strcmp(option_name, "verbosity") == 0) {
verbosity = atoi(option_value);
} else if (strcmp(option_name, "scoring") == 0) {
if (strcmp(option_value, "max-odds") == 0)
scoring = MAX_ODDS;
else if (strcmp(option_value, "avg-odds") == 0)
scoring = AVG_ODDS;
else if (strcmp(option_value, "sum-odds") == 0)
scoring = SUM_ODDS;
else
die("Specified scoring scheme not known.\n", message);
} else if (strcmp(option_name, "pvalues") == 0) {
pvalues = TRUE;
} else if (strcmp(option_name, "rma") == 0) {
normalize_scores = TRUE;
fprintf(stderr, "Normalizing motif scores using RMA method.\n");
} else if (strcmp(option_name, "gcbins") == 0) {
num_gc_bins = atoi(option_value);
pvalues = TRUE;
if (num_gc_bins <= 1) die("Number of bins in --gcbins must be greater than 1.\n", message);
} else if (strcmp(option_name, "sdbg") == 0) {
sdbg_order = atoi(option_value); // >=0 means use sequence bkg
}
else if (strcmp(option_name, "last") == 0) {
int i = 0;
if (option_value[0] == '-') ++i;
while (option_value[i] != '\0') {
if (!isdigit(option_value[i])) {
die("Specified parameter 'last' contains non-numeric characters.\n");
}
++i;
}
last = atoi(option_value);
if (errno != 0) {
die("Specified parameter 'last' could not be parsed as a number as:\n%s\n",strerror(errno));
}
if (last < 0) {
die("Specified parameter 'last' had negative value (%d) when only postive or zero values are allowed \n", last);
}
}
else if (strcmp(option_name, "version") == 0) {
fprintf(stdout, VERSION "\n");
exit(EXIT_SUCCESS);
}
}
// --sdbg overrides --pvalues and --gcbins and --rma
int req_args = 3;
if (sdbg_order >= 0) {
pvalues = FALSE;
normalize_scores = FALSE;
num_gc_bins = 1;
req_args = 2;
}
// Check all required arguments given
if (sdbg_order >= 0 && argc > option_index + req_args) {
die("<background file> cannot be given together with --sdbg.\n");
} else if (argc != option_index + req_args) {
fprintf(stderr, usage, pseudocount, verbosity, max_seq_length);
exit(EXIT_FAILURE);
}
// Get required arguments.
char* motif_filename = argv[option_index];
option_index++;
char* fasta_filename = argv[option_index];
option_index++;
char* bg_filename;
if (req_args == 3) { // required unless --sdbg given
bg_filename = argv[option_index];
option_index++;
} else {
bg_filename = "--uniform--"; // So PSSMs will use uniform background;
// we can multiply them out later.
}
// measure time
c0 = clock();
// Set up hash tables for computing reverse complement if doing --sdbg
if (sdbg_order >= 0) setup_hash_alph(DNAB);
// Create cisml data structure for recording results
cisml = allocate_cisml(program_name, motif_filename, fasta_filename);
set_cisml_background_file(cisml, bg_filename);
/**********************************************
* Read the motifs and background model.
**********************************************/
int num_motifs = 0;
MREAD_T *mread;
ARRAYLST_T *motifs;
PSSM_PAIR_T** pssm_pairs; // note pssm_pairs is an array of pointers
//this reads any meme file, xml, txt and html
mread = mread_create(motif_filename, OPEN_MFILE);
mread_set_bg_source(mread, bg_filename);
mread_set_pseudocount(mread, pseudocount);
motifs = mread_load(mread, NULL);
alph = mread_get_alphabet(mread);
pos_bg_freqs = mread_get_background(mread);
mread_destroy(mread);
num_motifs = arraylst_size(motifs);
// allocate memory for PSSM pairs
pssm_pairs = (PSSM_PAIR_T**)mm_malloc(sizeof(PSSM_PAIR_T*) * num_motifs);
if (verbosity >= NORMAL_VERBOSE)
fprintf(stderr, "Number of motifs in file %d.\n", num_motifs);
// make a CISML pattern to hold scores for each motif
PATTERN_T** patterns = NULL;
Resize(patterns, num_motifs, PATTERN_T*);
int motif_index;
for (motif_index = 0; motif_index < num_motifs; motif_index++) {
MOTIF_T* motif = (MOTIF_T*)arraylst_get(motif_index, motifs);
patterns[motif_index] = allocate_pattern(get_motif_id(motif), "");
add_cisml_pattern(cisml, patterns[motif_index]);
}
// make reverse complement motifs and background frequencies.
if (scan_both_strands == TRUE) {
add_reverse_complements(motifs);
assert(arraylst_size(motifs) == (2 * num_motifs));
rev_bg_freqs = allocate_array(get_array_length(pos_bg_freqs));
complement_dna_freqs(pos_bg_freqs, rev_bg_freqs);
}
/**************************************************************
* Convert motif matrices into log-odds matrices.
* Scale them.
* Compute the lookup tables for the PDF of scaled log-odds scores.
**************************************************************/
int ns = scan_both_strands ? 2 : 1; // number of strands
for (motif_index = 0; motif_index < num_motifs; motif_index++) {
MOTIF_T *motif, *motif_rc;
motif = (MOTIF_T*)arraylst_get(motif_index*ns, motifs);
if (scan_both_strands)
motif_rc = (MOTIF_T*)arraylst_get(motif_index*ns + 1, motifs);
else
motif_rc = NULL;
/*
* Note: If scanning both strands, we complement the motif frequencies
* but not the background frequencies so the motif looks the same.
* However, the given frequencies are used in computing the p-values
* since they represent the frequencies on the negative strands.
* (If we instead were to complement the input sequence, keeping the
* the motif fixed, we would need to use the complemented frequencies
* in computing the p-values. Is that any clearer?)
*/
double range = 300; // 100 is not very good; 1000 is great but too slow
PSSM_T* pos_pssm =
build_motif_pssm(
motif,
pos_bg_freqs,
pos_bg_freqs,
NULL, // Priors not used
0.0L, // alpha not used
range,
num_gc_bins,
TRUE
);
PSSM_T* neg_pssm = (scan_both_strands ?
build_motif_pssm(
motif_rc,
rev_bg_freqs,
pos_bg_freqs,
NULL, // Priors not used
0.0L, // alpha not used
range,
num_gc_bins,
TRUE
)
: NULL
);
pssm_pairs[motif_index] = create_pssm_pair(pos_pssm, neg_pssm);
}
// Open the FASTA file for reading.
FILE* fasta_file = NULL;
if (open_file(fasta_filename, "r", FALSE, "FASTA", "sequences", &fasta_file) == 0) {
die("Couldn't open the file %s.\n", fasta_filename);
}
if (verbosity >= NORMAL_VERBOSE) {
if (last == 0) {
fprintf(stderr, "Using entire sequence\n");
} else {
fprintf(stderr, "Limiting sequence to last %d positions.\n", last);
}
}
/**************************************************************
* Read in all sequences and score with all motifs
**************************************************************/
int seq_loading_num = 0; // keeps track on the number of sequences read in total
int seq_counter = 0; // holds the index to the seq in the pattern
int unique_seqs = 0; // keeps track on the number of unique sequences
BOOLEAN_T need_postprocessing = FALSE;
SEQ_T* sequence = NULL;
RBTREE_T* seq_ids = rbtree_create(rbtree_strcasecmp,NULL,free,rbtree_intcpy,free);
RBNODE_T* seq_node;
BOOLEAN_T created;
while (read_one_fasta(alph, fasta_file, max_seq_length, &sequence)) {
++seq_loading_num;
created = FALSE;
char* seq_name = get_seq_name(sequence);
int seq_len = get_seq_length(sequence);
int scan_len;
if (last != 0) {
scan_len = last;
} else {
scan_len = seq_len;
}
// red-black trees are only required if duplicates should be combined
if (combine_duplicates){
//lookup seq id and create new entry if required, return sequence index
char *tmp_id = mm_malloc(strlen(seq_name)+1); // required copy for rb-tree
strncpy(tmp_id,seq_name,strlen(seq_name)+1);
seq_node = rbtree_lookup(seq_ids, tmp_id, TRUE, &created);
if (created) {// assign it a loading number
rbtree_set(seq_ids, seq_node, &unique_seqs);
seq_counter = unique_seqs;
++unique_seqs;
} else {
seq_counter = *((int*)rbnode_get(seq_node));
}
}
//
// Set up sequence-dependent background model and compute
// log cumulative probability of sequence.
//
double *logcumback = NULL; // array of log cumulative probs.
if (sdbg_order >= 0) {
Resize(logcumback, seq_len+1, double);
char* raw_seq = get_raw_sequence(sequence);
BOOLEAN rc = FALSE;
double *a_cp = get_markov_from_sequence(raw_seq, alph_string(alph), rc, sdbg_order, 0);
log_cum_back(raw_seq, a_cp, sdbg_order, logcumback);
myfree(a_cp);
}
// Get the GC content of the sequence if binning p-values by GC
// and store it in the sequence object.
if (num_gc_bins > 1) {
ARRAY_T *freqs = get_sequence_freqs(sequence, alph);
set_total_gc_sequence(sequence,
get_array_item(1,freqs) + get_array_item(2,freqs)); // f(C) + f(G)
free_array(freqs); // clean up
} else {
set_total_gc_sequence(sequence, -1); // flag ignore
}
/**************************************************************
* Process all motifs.
**************************************************************/
int ns = scan_both_strands ? 2 : 1;
for (motif_index = 0; motif_index < num_motifs; motif_index++) {
PATTERN_T *pattern = patterns[motif_index];
MOTIF_T* motif = (MOTIF_T*)arraylst_get(ns*motif_index, motifs);
char* motif_id = (scan_both_strands ? get_motif_st_id(motif) : get_motif_id(motif));
if (verbosity >= HIGH_VERBOSE) {
fprintf(stderr, "Using motif %s of width %d.\n", motif_id, get_motif_length(motif));
}
if ((selected_motifs == NULL) || (have_string(get_motif_id(motif), selected_motifs) == TRUE)) {
if (verbosity >= HIGHER_VERBOSE) {
fprintf(stderr, "Scanning %s sequence with length %d "
"abbreviated to %d with motif %s with length %d.\n",
seq_name, seq_len, scan_len, motif_id, get_motif_length(motif));
}
SCANNED_SEQUENCE_T* scanned_seq = NULL;
if (!combine_duplicates || get_pattern_num_scanned_sequences(pattern) <= seq_counter){
// Create a scanned_sequence record and save it in the pattern.
scanned_seq = allocate_scanned_sequence(seq_name, seq_name, pattern);
set_scanned_sequence_length(scanned_seq, scan_len);
} else {
// get existing sequence record
scanned_seq = get_pattern_scanned_sequences(pattern)[seq_counter];
set_scanned_sequence_length(scanned_seq, max(scan_len, get_scanned_sequence_length(scanned_seq)));
}
// check if scanned component of sequence has sufficient length for the motif
if (scan_len < get_motif_length(motif)) {
// set score to zero and p-value to 1 if not set yet
if(!has_scanned_sequence_score(scanned_seq)){
set_scanned_sequence_score(scanned_seq, 0.0);
}
if(pvalues && !has_scanned_sequence_pvalue(scanned_seq)){
set_scanned_sequence_pvalue(scanned_seq, 1.0);
}
add_scanned_sequence_scanned_position(scanned_seq);
if (get_scanned_sequence_num_scanned_positions(scanned_seq) > 0L) need_postprocessing = TRUE;
if (verbosity >= HIGH_VERBOSE) fprintf(stderr, "%s too short for motif %s. Score set to 0!\n", seq_name, motif_id);
} else {
// scan the sequence using average/maximum motif affinity
ama_sequence_scan(alph, sequence, logcumback, pssm_pairs[motif_index], scoring,
pvalues, last, scanned_seq, &need_postprocessing);
}
} else {
if (verbosity >= HIGH_VERBOSE) fprintf(stderr, "Skipping motif %s.\n", motif_id);
}
} // All motifs parsed
free_seq(sequence);
if (sdbg_order >= 0) myfree(logcumback);
} // read sequences
/*************************************************************************
* Calculate the odds score for each motif-sized window at each
* site in the sequence using the given nucleotide frequencies.
*
* This function is a lightweight version based on the one contained in
* motiph-scoring. Several calculations that are unnecessary for gomo
* have been removed in order to speed up the process.
* Scores sequence with up to two motifs.
*************************************************************************/
double score_sequence(
SEQ_T* seq, // sequence to scan (IN)
double *logcumback, // cumulative bkg probability of sequence (IN)
PSSM_PAIR_T* pssm_pair, // pos and neg pssms (IN)
int method, // method used for scoring (IN)
int last, //score only last <n> or
//score all if <n> is zero (IN)
BOOLEAN_T* isFeasible // FLAG indicated if there is at least one position
// where the motif could be matched against (OUT)
)
{
assert(pssm_pair != NULL);
assert(seq != NULL);
PSSM_T* pos_pssm = pssm_pair->pos_pssm;
assert(pos_pssm != NULL);
PSSM_T* neg_pssm = pssm_pair->neg_pssm;
int n_motifs = neg_pssm ? 2 : 1;
char* raw_seq = get_raw_sequence(seq);
int seq_length = get_seq_length(seq);
int w = get_num_rows(pos_pssm->matrix);
int n = seq_length - w + 1;
if (verbosity >= DUMP_VERBOSE) {
fprintf(stderr, "Debug n_motifs: %d seq_length: %d w: %d n: %d.\n", n_motifs, seq_length, w, n);
}
// Get alphabet;
char* alphabet = get_alphabet(FALSE);
int alph_size = get_alph_size(ALPH_SIZE);
// Dependent on the "last" parameter, change the starting point
int start;
int N_scored;
if (last > 0 && last < seq_length) {
start = seq_length - last;
N_scored = n_motifs * (last - w + 1); // number of sites scored
} else {
start = 0;
N_scored = n_motifs * n; // number of sites scored
}
// For each motif (positive and reverse complement)
double max_odds = 0.0;
double sum_odds = 0.0;
double requested_odds = 0.0;
int i;
if (verbosity >= HIGHER_VERBOSE) {
fprintf(stderr, "Starting scan at position %d .\n", start);
}
for (i=0; i<n_motifs; i++) { // pos (and negative) motif
PSSM_T* pssm = (i==0 ? pos_pssm : neg_pssm); // choose +/- motif
// For each site in the sequence
int seq_index;
for (seq_index = start; seq_index < n; seq_index++) { // site
double odds = 1.0;
// For each position in the motif window
int motif_position;
for (motif_position = 0; motif_position < w; motif_position++) { // column
int i_site = seq_index + motif_position;
char c = raw_seq[i_site];
// Check for gaps at this site
if (c == '-' || c == '.') { N_scored--; odds = 0; break; }
// Check for ambiguity codes at this site
int alph_index = alphabet_index(c, alphabet);
if (alph_index >= alph_size || alph_index < 0) { N_scored--; odds = 0; break; }
// multiple odds by value in appropriate motif cell
odds *= get_matrix_cell(motif_position, alph_index, pssm->matrix);
} // column
//
// Apply sequence-dependent background model.
//
if (logcumback) {
int i_site = seq_index;
double log_p = logcumback[i_site+w] - logcumback[i_site]; // log Pr(x | background)
//printf("log_p:: %g motif_pos %d\n", log_p, motif_position);
double adjust = exp(w*log(1/4.0) - log_p); // Pr(x | uniform) / Pr(x | background)
odds *= adjust;
}
// Add odds to growing sum.
sum_odds += odds; // sum of odds
if (odds > max_odds) max_odds = odds; // max of odds
} // site
} // motif
if (verbosity >= HIGHER_VERBOSE) {
fprintf(stderr, "Scored %d positions with the sum odds %f and the max odds %f.\n", N_scored, sum_odds, max_odds);
}
// has there been anything matched at all?
if (N_scored == 0){
if (verbosity >= NORMAL_VERBOSE) {
fprintf(stderr,"Sequence \'%s\' offers no location to match the motif against (sequence length too short?)\n",get_seq_name(seq));
}
*isFeasible = FALSE;
return 0.0;
// return odds as requested (MAX or AVG scoring)
} else if (method == AVG_ODDS) {
requested_odds = sum_odds / N_scored; // mean
} else if (method == MAX_ODDS) {
requested_odds = max_odds; // maximum
} else if (method == SUM_ODDS) {
requested_odds = sum_odds ; // sum
}
return(requested_odds);
} // score_sequence
/*************************************************************************
* Calculate the log-odds score for each possible motif site in the
* sequence and record the sites of the best. Apply a count to each
* best site and increment the total site count.
*************************************************************************/
static void score_sequence(
CENTRIMO_OPTIONS_T *options,
SEQ_T* sequence,
PSSM_T* pssm,
PSSM_T* rev_pssm,
SEQ_SITES_T* seq_sites,
SITE_COUNTS_T* counts
)
{
char *raw_seq, *seg;
int i, L, w, pos;
double score;
double count;
SEQ_SITE_T *site;
// check we got passed stuff
assert(options != NULL);
assert(sequence != NULL);
assert(pssm != NULL);
assert(seq_sites != NULL);
assert(counts != NULL);
// make Mac OS compiler happy.
score = -BIG;
// Score and record each possible motif site in the sequence
raw_seq = get_raw_sequence(sequence);
L = get_seq_length(sequence);
w = pssm->w;
// Reset the sequence stats structure
seq_sites->best = -BIG;
seq_sites->used = 0;
// Read and score each position in the sequence.
for (i = 0; i < L - w + 1; i++) {
seg = raw_seq+i;
// Score and record forward strand
if (score_motif_site(options->alphabet, seg, pssm, &score))
track_site(seq_sites, score, i, '+');
// Score and record reverse strand if appropriate.
if (rev_pssm && score_motif_site(options->alphabet, seg, rev_pssm, &score))
track_site(seq_sites, score, i, '-');
}
// Record the position of best site, averaging ties
// and using position in RC of sequence if site on reverse strand
// unless no_flip is true.
if (seq_sites->used && seq_sites->best >= options->score_thresh) {
// add 1/n_ties to each tied position's count,
// averaging rather than random choice
count = (double)1.0 / (double)seq_sites->used;
for (i = 0; i < seq_sites->used; i++) {
site = seq_sites->sites+i;
if (options->no_flip || site->strand == '+') {
//pos = 2 * (site->start + w/2 - 1/2); // a motif of width 1 can have sites at the first index
pos = 2 * site->start + w - 1; // a motif of width 1 can have sites at the first index
} else {
//pos = 2 * (L - (site->start + w/2) - 1; // a motif of width 1 can have sites at the first index
pos = 2 * (L - site->start) - w - 1;
}
//record the count
counts->sites[pos] += count;
}
counts->total_sites++;
}
}
/****************************************************************************
* Allocate one alignment object. Name and description may be NULL.
*
* Returns a pointer to the newly created alignment.
****************************************************************************/
ALIGNMENT_T* allocate_alignment(
char* name,
char* description,
int num_sequences,
SEQ_T** sequences,
char* consensus_string
)
{
assert(num_sequences > 0);
assert(sequences != NULL);
assert(consensus_string != NULL);
// Allocate the alignment object.
ALIGNMENT_T* new_alignment = (ALIGNMENT_T*)mm_malloc(sizeof(ALIGNMENT_T));
if (new_alignment == NULL) {
die("Error allocating alignment\n");
}
// Store the name, truncating if necessary.
if (name != NULL) {
strncpy(new_alignment->name, name, MAX_ALIGNMENT_NAME);
new_alignment->name[MAX_ALIGNMENT_NAME] = '\0';
if (strlen(new_alignment->name) != strlen(name)) {
fprintf(stderr, "Warning: truncating alignment program name %s to %s.\n",
name, new_alignment->name);
}
} else {
new_alignment->name[0] = '\0';
}
// Store the description, truncating if necessary.
if (description != NULL) {
strncpy(new_alignment->desc, description, MAX_ALIGNMENT_COMMENT);
new_alignment->desc[MAX_ALIGNMENT_COMMENT] = '\0';
} else {
new_alignment->desc[0] = '\0';
}
// Store the sequences.
new_alignment->sequences = (SEQ_T**) mm_malloc(num_sequences * sizeof(SEQ_T*));
if (new_alignment->sequences == NULL) {
die("Error allocating sequences\n");
}
new_alignment->num_sequences = num_sequences;
int seq_length = strlen(get_raw_sequence(sequences[0]));
int i;
for (i = 0; i < num_sequences; i++) {
myassert(TRUE,
strlen(get_raw_sequence(sequences[i])) == seq_length,
"Sequence #1 (%s) is length=%d, but sequence #%d (%s) is length=%d.\n<%s>\n",
get_seq_name(sequences[0]), seq_length, i,
get_seq_name(sequences[i]), strlen(get_raw_sequence(sequences[i])),
get_raw_sequence(sequences[i]));
new_alignment->sequences[i] =
allocate_seq(get_seq_name(sequences[i]),
get_seq_description(sequences[i]),
get_seq_offset(sequences[i]),
get_raw_sequence(sequences[i])
);
}
// Fill in the remaining fields.
new_alignment->length = seq_length;
copy_string(&(new_alignment->consensus_string), consensus_string);
return(new_alignment);
}
