main(int argc, char **argv) {
char *aa0;
char libstr[MAX_FN];
char qname[MAX_FN];
int sq0off;
int i, n0;
FILE *fp;
struct pstruct pst, *ppst;
/* stuff from initfa.c/h_init() */
memcpy(qascii,aascii,sizeof(qascii));
/* initialize a pam matrix */
ppst = &pst;
strncpy(ppst->pamfile,"BL50",MAX_FN);
standard_pam(ppst->pamfile,ppst,0,0);
/* this is always protein by default */
ppst->nsq = naa;
ppst->nsqx = naax;
for (i=0; i<=ppst->nsqx; i++) {
ppst->sq[i] = aa[i];
ppst->hsq[i] = haa[i];
ppst->sqx[i]=aax[i]; /* sq = aa */
ppst->hsqx[i]=haax[i]; /* hsq = haa */
}
ppst->sq[ppst->nsqx+1] = ppst->sqx[ppst->nsqx+1] = '\0';
if ((aa0 = calloc(MAXTST,sizeof(char)))==NULL) {
fprintf(stderr,"Cannot allocate aa0\n");
exit(1);
}
initenv(argc, argv, &pst, qname);
alloc_pam(pst.nsq+1,pst.nsq+1, &pst);
initpam2(&pst);
n0 = getseq (qname, qascii, aa0, MAXTST, libstr,&sq0off);
if (!pst.pam_pssm) {
fprintf(stderr," ** ERROR ** No -P PSSM provided\n");
}
else {
ppst->pam2p[0] = alloc_pam2p(n0,pst.nsq);
ppst->pam2p[1] = alloc_pam2p(n0,pst.nsq);
if ((fp = fopen(pst.pgpfile,"rb"))!=NULL) {
read_pssm(aa0, n0, pst.nsq, pst.pamscale,fp,ppst);
}
}
}
int main( int argc, char* argv[] ) {
char mat_fn[MAX_FN_LEN+1];
char maln_fn[MAX_FN_LEN+1];
char fastq_out_fn[MAX_FN_LEN+1];
char maln_root[MAX_FN_LEN+1];
char ref_fn[MAX_FN_LEN+1];
char frag_fn[MAX_FN_LEN+1];
char adapter_code[2]; // place to keep the argument for -a (which adapter to trim)
char* c_time; // place to keep asctime string
char* test_id;
int ich;
int any_arg = 0;
int Hard_cut = 0; // If 0 => use dynamic score cutoff, if > 0 use this instead
int circular = 0; // Boolean, TRUE if reference sequence is circular
int make_fastq = 0; // Boolean, TRUE if we should also output fastq database of seqs in assembly
int seq_code = 0; // code to indicate sequence input format; 0 => fasta; 1 => fastq
int do_adapter_trimming = 0; // Boolean, TRUE if we should try to trim
// adapter from input sequences
int iterate = 0; //Boolean, TRUE means interate the assembly until convergence
// on an assembled sequence
int FINAL_ONLY = 0; //Boolean, TRUE means only write out the final assembly maln file
// FALSE (default) means write out each one
int ids_rest = 0; // Boolean, TRUE means restrict analysis to IDs in input file
int repeat_filt = 0; //Boolean, TRUE means remove sequences that are repeats,
// keeping best-scoring representative
int repeat_qual_filt = 0; //Boolean, TRUE means remove sequences that are repeats,
// keeping best quality score sum representative
int just_outer_coords = 1; // Boolean, TRUE means just use strand, start, and end to
// determine if sequences are redundant
int SCORE_CUT_SET = 0; //Boolean, TRUE means user has set a length/score cutoff line
int seen_seqs = 0;
int hp_special = 0; // Boolean, TRUE means user wants hp gap special discount
int distant_ref = 0; // Boolean, TRUE means the initial reference sequence is
// known to be distantly related so keep trying to align all
// sequences each round
int kmer_filt_len = -1; // length of kmer filtering, if user wants it; otherwise
// special value of -1 indicates this is unset
int soft_mask = 0; //Boolean; TRUE => do not use kmers that are all lower-case
// FALSE => DO use all kmers, regardless of case
int iter_num; // Number of iterations of assembly done
int collapse = 0; // Boolean; TRUE => collapse input sequences in FSDB to improve
// sequence quality
// FALSE => (default) keep all sequences
double slope = DEF_S; // Set these to default unless, until user changes
double intercept = DEF_N; // them
MapAlignmentP maln, // Contains all fragments initially better
// than FIRST_ROUND_SCORE_CUTOFF
culled_maln; // Contains all fragments with scores
// better than SCORE_CUTOFF
AlignmentP fw_align, rc_align, adapt_align;
PSSMP ancsubmat = init_flatsubmat();
PSSMP rcancsubmat = revcom_submat(ancsubmat);
const PSSMP flatsubmat = init_flatsubmat();
KPL* fkpa; // Place to keep forward kmer array if user requested kmer
KPL* rkpa; // Place to keep reverse kmer array if user requested kmer
IDsListP good_ids;
FragSeqP frag_seq;
PWAlnFragP front_pwaln, back_pwaln;
FSDB fsdb; // Database to hold sequences to iterate over
FILE* FF;
time_t curr_time;
char maln_root_def[] = "assembly.maln.iter";
extern int optind;
extern char* optarg;
char neand_adapt[] = "GTCAGACACGCAACAGGGGATAGGCAAGGCACACAGGGGATAGG";
char stand_adapt[] = "CTGAGACACGCAACAGGGGATAGGCAAGGCACACAGGGGATAGG";
char user_def_adapt[128];
char* adapter; // set to either neand_adapt or stand_adapt based on user preference
adapter = neand_adapt; // Default is Neandertal
char* assembly_cons;
char* last_assembly_cons;
int cc = 1; // consensus code for calling consensus base
int i;
/* Set the default output filename until the user overrides it */
strcpy( maln_root, maln_root_def );
/* Process command line arguments */
while( (ich=getopt( argc, argv, "s:r:f:m:a:p:H:I:S:N:k:q:FTciuhDMUAC" )) != -1 ) {
switch(ich) {
case 'c' :
circular = 1;
break;
case 'q' :
make_fastq = 1;
strcpy( fastq_out_fn, optarg );
case 'C' :
collapse = 1;
break;
case 'i' :
iterate = 1;
break;
case 'h' :
hp_special = 1;
break;
case 'u' :
repeat_filt = 1;
break;
case 'A' :
just_outer_coords = 0;
break;
case 'U' :
repeat_qual_filt = 1;
break;
case 'D' :
distant_ref = 1;
break;
case 'p' :
cc = atoi( optarg );
any_arg = 1;
break;
case 'I' :
good_ids = parse_ids( optarg );
ids_rest = 1;
break;
case 'H' :
Hard_cut = atoi( optarg );
if ( Hard_cut <= 0 ) {
fprintf( stderr, "Hard cutoff (-H) must be positive\n" );
help();
exit( 0 );
}
any_arg = 1;
break;
case 'M' :
soft_mask = 1;
break;
case 's' :
strcpy( mat_fn, optarg );
free( ancsubmat ); // trash the flat submat we initialized with
ancsubmat = read_pssm( mat_fn );
free( rcancsubmat ); // trash the init rcsubmat, too
rcancsubmat = revcom_submat( ancsubmat );
any_arg = 1;
break;
case 'r' :
strcpy( ref_fn, optarg );
any_arg = 1;
break;
case 'k' :
kmer_filt_len = atoi( optarg );
any_arg = 1;
break;
case 'f' :
strcpy( frag_fn, optarg );
any_arg = 1;
break;
case 'm' :
strcpy( maln_root, optarg );
any_arg = 1;
break;
case 'T' :
do_adapter_trimming = 1;
break;
case 'a' :
if ( strlen( optarg ) > 127 ) {
fprintf( stderr, "That adapter is too big!\nMIA will use the standard adapter.\n" );
adapter = stand_adapt;
}
else {
strcpy( user_def_adapt, optarg );
if ( strlen( user_def_adapt ) > 1 ) {
adapter = user_def_adapt;
}
else {
if ( !( (user_def_adapt[0] == 'n') ||
(user_def_adapt[0] == 'N') ) ) {
adapter = stand_adapt;
}
else {
adapter = neand_adapt;
}
}
}
break;
case 'S' :
slope = atof( optarg );
SCORE_CUT_SET = 1;
break;
case 'N' :
intercept = atof( optarg );
SCORE_CUT_SET = 1;
break;
case 'F' :
FINAL_ONLY = 1;
break;
default :
help();
exit( 0 );
}
}
if ( !any_arg ) {
help();
exit( 0 );
}
if ( optind != argc ) {
fprintf( stderr, "There seems to be some extra cruff on the command line that mia does not understand.\n" );
}
/* Start the clock... */
curr_time = time(NULL);
// c_time = (char*)save_malloc(64*sizeof(char));
// c_time = asctime(localtime(&curr_time));
/* Announce that we're starting */
fprintf( stderr,
"Starting assembly of %s\nusing %s\nas reference at %s\n",
frag_fn, ref_fn,
asctime(localtime(&curr_time)) );
/* Set up the maln structure */
maln = (MapAlignmentP)init_map_alignment();
maln->cons_code = cc;
if ( maln == NULL ) {
fprintf( stderr, "Not enough memories for this\n" );
exit( 1 );
}
/* Set the distant_ref flag */
maln->distant_ref = distant_ref;
/* Set up the FSDB for keeping good-scoring sequence in memory */
fsdb = init_FSDB();
if ( fsdb == NULL ) {
fprintf( stderr, "Not enough memories for holding sequences\n" );
exit( 1 );
}
/* Read in the reference sequence and make reverse complement, too*/
if ( read_fasta_ref( maln->ref, ref_fn ) != 1 ) {
fprintf( stderr, "Problem reading reference sequence file %s\n", ref_fn );
exit( 1 );
}
/* Add wrap-around sequence (rc, too) and set maln->ref->circular
if it's circular */
if ( circular ) {
add_ref_wrap( maln->ref );
}
else {
maln->ref->wrap_seq_len = maln->ref->seq_len;
}
/* Add space for the gaps array */
maln->ref->gaps = (int*)save_malloc((maln->ref->wrap_seq_len+1) *
sizeof(int));
for( i = 0; i <= maln->ref->wrap_seq_len; i++ ) {
maln->ref->gaps[i] = 0;
}
/* Set up fkpa and rkpa for list of kmers in the reference (forward and
revcom strand) if user wants kmer filtering */
if ( kmer_filt_len > 0 ) {
fprintf( stderr, "Making kmer list for k-mer filtering...\n" );
fkpa = init_kpa(kmer_filt_len);
rkpa = init_kpa(kmer_filt_len);
/*
kmer_list = (KmersP)pop_kmers( maln->ref, kmer_filt_len );
*/
populate_kpa( fkpa, maln->ref->seq,
maln->ref->wrap_seq_len, kmer_filt_len,
soft_mask );
populate_kpa( rkpa, maln->ref->rcseq,
maln->ref->wrap_seq_len, kmer_filt_len,
soft_mask );
}
/* Now kmer arrays have been made if requested. We can upper case
the reference sequences. */
make_ref_upper( maln->ref );
/* Set up FragSeqP to point to a FragSeq */
frag_seq = (FragSeqP)save_malloc(sizeof(FragSeq));
/* Set up the alignment structures for forward and reverse
complement alignments */
fw_align = (AlignmentP)init_alignment( INIT_ALN_SEQ_LEN,
(maln->ref->wrap_seq_len +
(2*INIT_ALN_SEQ_LEN)),
0, hp_special );
rc_align = (AlignmentP)init_alignment( INIT_ALN_SEQ_LEN,
(maln->ref->wrap_seq_len +
(2*INIT_ALN_SEQ_LEN)),
1, hp_special );
/* Set up the alignment structure for adapter trimming, if user
wants that */
if ( do_adapter_trimming ) {
adapt_align = (AlignmentP)init_alignment( INIT_ALN_SEQ_LEN,
INIT_ALN_SEQ_LEN,
0, hp_special );
/* Setup the flatsubmat */
//flatsubmat = init_flatsubmat();
adapt_align->submat = flatsubmat;
adapt_align->seq2 = adapter;
adapt_align->len2 = strlen( adapt_align->seq2 );
pop_s2c_in_a( adapt_align );
if ( hp_special ) {
pop_hpl_and_hps( adapt_align->seq2, adapt_align->len2,
adapt_align->hprl, adapt_align->hprs );
}
/* Set for a semi-global that pays a penalty for unaligning the
beginning of the adapter, but not for the end of the adapter.
This is because if the sequence read (align->seq1) ends, then
we won't see any more of the adapter. When we search for the
best alignment, we'll only look in the last column, requiring that
all of align->seq1 is accounted for */
adapt_align->sg5 = 1;
adapt_align->sg3 = 0;
}
fw_align->seq1 = maln->ref->seq;
rc_align->seq1 = maln->ref->rcseq;
if ( circular ) {
fw_align->len1 = maln->ref->wrap_seq_len;
rc_align->len1 = maln->ref->wrap_seq_len;
}
else {
fw_align->len1 = maln->ref->seq_len;
rc_align->len1 = maln->ref->seq_len;
}
/* Now the reference sequence and its reverse complement are
prepared, put the s1c lookup codes in */
pop_s1c_in_a( fw_align );
pop_s1c_in_a( rc_align );
if ( hp_special ) {
pop_hpl_and_hps( fw_align->seq1, fw_align->len1,
fw_align->hpcl, fw_align->hpcs );
pop_hpl_and_hps( rc_align->seq1, rc_align->len1,
rc_align->hpcl, rc_align->hpcs );
}
/* One by one, go through the input file of fragments to be aligned.
Align them to the reference. For each fragment generating an
alignment score better than the cutoff, merge it into the maln
alignment. Keep track of those that don't, too. */
FF = fileOpen( frag_fn, "r" );
seq_code = find_input_type( FF );
//LOG = fileOpen( log_fn, "w" );
front_pwaln = (PWAlnFragP)save_malloc( sizeof(PWAlnFrag));
back_pwaln = (PWAlnFragP)save_malloc( sizeof(PWAlnFrag));
/* Give some space to remember the IDs as we see them */
test_id = (char*)save_malloc(MAX_ID_LEN * sizeof(char));
/* Announce we're strarting alignment of fragments */
fprintf( stderr, "Starting to align sequences to the reference...\n" );
while( read_next_seq( FF, frag_seq, seq_code ) ) {
seen_seqs++;
strcpy( test_id, frag_seq->id );
if ( DEBUG ) {
fprintf( stderr, "%s\n", frag_seq->id );
}
if ( !ids_rest ||
( bsearch( &test_id, good_ids->ids,
good_ids->num_ids,
sizeof(char*), idCmp )
!= NULL ) ) {
if ( do_adapter_trimming ) {
/* Trim sequence (set frag_seg->trimmed and
frag_seg->trim_point field) */
trim_frag( frag_seq, adapter, adapt_align );
}
else {
frag_seq->trimmed = 0;
}
/* Check if kmer filtering. If so, filter */
if ( new_kmer_filter( frag_seq, fkpa, rkpa, kmer_filt_len,
fw_align, rc_align ) ) {
/* Align this fragment to the reference and write
the result into pwaln; use the ancsubmat, not the reverse
complemented rcsancsubmat during this first iteration because
all sequence is forward strand
*/
fw_align->submat = ancsubmat;
rc_align->submat = ancsubmat;
if ( sg_align( maln, frag_seq, fsdb,
fw_align, rc_align,
front_pwaln,
back_pwaln ) == 0 ) {
fprintf( stderr, "Problem handling %s\n", frag_seq->id );
}
}
}
if ( seen_seqs % 1000 == 0 ) {
fprintf( stderr, "." );
}
if ( seen_seqs % 80000 == 0 ) {
fprintf( stderr, "\n" );
}
}
/* Now, fsdb is complete and points to all the things in maln.
So we can fill in the AlnSeqP->smp array for everything in the
maln->AlnSeqArray to know which matrices to use for *CALLING*
a consensus; Conveniently, there are pointers to all of these
in the fss->fss[X]->front|back_asp */
pop_smp_from_FSDB( fsdb, PSSM_DEPTH );
//fprintf( LOG, "__Finished with initial alignments__" );
//fflush( LOG );
fprintf( stderr, "\n" );
iter_num = 1;
/* Now, we need a new MapAlignment, culled_maln, that is big
enough to hold all the unique guys from maln */
culled_maln = init_culled_map_alignment( maln );
/* Filtering repeats announcement */
fprintf( stderr, "Repeat and score filtering\n" );
/* If user wants to filter against repeats by alignment score, do it */
if ( repeat_filt ) {
/* Sort fsdb by fsdb->as */
sort_fsdb( fsdb );
/* Now, everything is sorted in fsdb, so I can easily see
which guys are unique by as, ae, and rc fields */
set_uniq_in_fsdb( fsdb, just_outer_coords );
}
/* If user wants to filter against repeats by q-score sum, do it */
if ( repeat_qual_filt ) {
/* Sort fsdb by fsdb->as */
sort_fsdb_qscore( fsdb );
/* Now, everything is sorted in fsdb, so I can easily see
which guys are unique by as, ae, and rc fields */
set_uniq_in_fsdb( fsdb, just_outer_coords );
}
/* Now, we know which sequences are unique, so make a
culled_maln with just the unique guys */
cull_maln_from_fsdb( culled_maln, fsdb, Hard_cut,
SCORE_CUT_SET, slope, intercept );
fclose(FF);
/* Tell the culled_maln which matrices to use for assembly */
culled_maln->fpsm = ancsubmat;
culled_maln->rpsm = rcancsubmat;
sort_aln_frags( culled_maln ); //invalidates fsdb->front|back_asp fields!
fw_align->submat = ancsubmat;
fw_align->sg5 = 1;
fw_align->sg3 = 1;
last_assembly_cons = (char*)save_malloc((maln->ref->seq_len +1) *
sizeof(char));
strncpy( last_assembly_cons, maln->ref->seq,
maln->ref->seq_len );
last_assembly_cons[maln->ref->seq_len] = '\0';
/* Re-align everything with revcomped
sequence and substitution matrices, but first
unmask all alignment positions and collapse sequences
if requested
*/
memset(fw_align->align_mask, 1, fw_align->len1);
if ( collapse ) {
collapse_FSDB( fsdb, Hard_cut, SCORE_CUT_SET,
slope, intercept );
}
reiterate_assembly( last_assembly_cons, iter_num, maln, fsdb,
fw_align, front_pwaln, back_pwaln,
ancsubmat, rcancsubmat );
pop_smp_from_FSDB( fsdb, PSSM_DEPTH );
fprintf( stderr, "Repeat and score filtering\n" );
if ( repeat_filt ) {
sort_fsdb( fsdb );
set_uniq_in_fsdb( fsdb, just_outer_coords );
}
if ( repeat_qual_filt ) {
sort_fsdb_qscore( fsdb );
set_uniq_in_fsdb( fsdb, just_outer_coords );
}
cull_maln_from_fsdb( culled_maln, fsdb, Hard_cut,
SCORE_CUT_SET, slope, intercept );
/* Tell the culled_maln which matrices to use for assembly */
culled_maln->fpsm = ancsubmat;
culled_maln->rpsm = rcancsubmat;
//invalidates fsdb->front|back_asp fields!
sort_aln_frags( culled_maln );
sprintf( maln_fn, "%s.%d", maln_root, iter_num );
if ( !iterate || !FINAL_ONLY ) {
write_ma( maln_fn, culled_maln );
if ( make_fastq ) {
write_fastq( fastq_out_fn, fsdb );
}
}
/* Are we iterating (re-aligning to the a new consensus? */
if (iterate) {
/* New assembly consensus announcement */
fprintf( stderr, "Generating new assembly consensus\n" );
assembly_cons = consensus_assembly_string( culled_maln );
while( ( strcmp( assembly_cons, last_assembly_cons ) != 0) &&
(iter_num < MAX_ITER) ) {
/* Another round...*/
iter_num++;
free( last_assembly_cons );
last_assembly_cons = assembly_cons;
fprintf( stderr, "Starting assembly iteration %d\n",
iter_num );
/* If the user wants collapsed sequences, now is the time */
if ( collapse ) {
collapse_FSDB( fsdb, Hard_cut, SCORE_CUT_SET,
slope, intercept );
}
reiterate_assembly( assembly_cons, iter_num, maln, fsdb,
fw_align, front_pwaln, back_pwaln,
ancsubmat, rcancsubmat );
pop_smp_from_FSDB( fsdb, PSSM_DEPTH );
fprintf( stderr, "Repeat and score filtering\n" );
if ( repeat_filt ) {
sort_fsdb( fsdb );
set_uniq_in_fsdb( fsdb, just_outer_coords );
}
if ( repeat_qual_filt ) {
sort_fsdb_qscore( fsdb );
set_uniq_in_fsdb( fsdb, just_outer_coords );
}
cull_maln_from_fsdb( culled_maln, fsdb, Hard_cut,
SCORE_CUT_SET, slope, intercept );
/* Tell the culled_maln which matrices to use for assembly */
culled_maln->fpsm = ancsubmat;
culled_maln->rpsm = rcancsubmat;
//invalidates fsdb->front|back_asp fields!
sort_aln_frags( culled_maln );
sprintf( maln_fn, "%s.%d", maln_root, iter_num );
if ( !FINAL_ONLY ) {
fprintf( stderr, "Writing maln file for iteration %d\n",
iter_num );
write_ma( maln_fn, culled_maln );
}
assembly_cons = consensus_assembly_string( culled_maln );
}
/* Convergence? */
if ( strcmp( assembly_cons, last_assembly_cons ) == 0 ) {
fprintf( stderr, "Assembly convergence - writing final maln\n" );
write_ma( maln_fn, culled_maln );
}
else {
fprintf( stderr, "Assembly did not converge after % rounds, quitting\n" );
write_ma( maln_fn, culled_maln );
}
if ( make_fastq ) {
write_fastq( fastq_out_fn, fsdb );
}
}
/* No iteration, but we must still re-align everything with revcomped
sequence and substitution matrices to keep scores comparable to what
they would have been had we iterated */
/* Announce we're finished */
curr_time = time(NULL);
// c_time = asctime(localtime(&curr_time));
fprintf( stderr, "Assembly finished at %s\n",
asctime(localtime(&curr_time)) );
exit( 0 );
}