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#include <cstdio>
#include <cstring>
#include <cstdlib>
#include <cassert>
#include <string>
#include <set>
#include <algorithm>
#include <stdint.h>
#include "htslib/sam.h"
#include "sam_utils.h"
#include "utils.h"
#include "my_assert.h"
using namespace std;
samFile *in;
bam_hdr_t *header;
bam1_t *b, *b2;
set<string> used;
bool isValid;
bool check_read(bam1_t *b, bam_hdr_t *header) {
uint32_t* cigar = bam_get_cigar(b);
for (int i = 0; i < b->core.n_cigar; ++i) {
char op = bam_cigar_opchr(*cigar);
if (op == 'N') {
printf("\nSkipped region is detected (cigar N) for read %s!\nTo use RSEM, please align your reads to a set of transcript sequences instead of a genome.\n", bam_get_qname(b));
return false;
}
else if (op == 'I' || op == 'D') {
printf("\nIndel alignment is detected (cigar %c) for read %s!\nRSEM currently does not support indel alignments.\n", op, bam_get_qname(b));
return false;
}
else if (op == 'S' || op == 'H' || op == 'P') {
printf("\nClipping or padding is detected (cigar %c) for read %s!\nRSEM currently doest not support clipping or padding.\n", op, bam_get_qname(b));
return false;
}
++cigar;
}
if (b->core.pos < 0 || bam_endpos(b) > header->target_len[b->core.tid]) {
printf("\n");
if (bam_is_paired(b)) {
printf("Mate %d of paired-end read %s", (bam_is_read1(b) ? 1 : 2), bam_get_qname(b));
}
else {
printf("Read %s", bam_get_qname(b));
}
printf(" aligns to [%d, %d) of transcript %s, which exceeds the transcript's boundary [0, %d)!\n",
b->core.pos, bam_endpos(b), header->target_name[b->core.tid], header->target_len[b->core.tid]);
return false;
}
return true;
}
int main(int argc, char* argv[]) {
if (argc != 2) {
printf("Usage: rsem-sam-validator <input.sam/input.bam/input.cram>\n");
exit(-1);
}
in = sam_open(argv[1], "r");
general_assert(in != 0, "Cannot open input file!");
header = sam_hdr_read(in);
general_assert(header != 0, "Cannot load SAM header!");
used.clear();
b = bam_init1(); b2 = bam_init1();
isValid = true;
HIT_INT_TYPE cnt = 0;
string cqname(""), qname;
int creadlen = 0, readlen, creadlen2, readlen2;
char ispaired = -1;
printf("."); fflush(stdout);
do {
int ret = sam_read1(in, header, b);
if (ret == -1) break;
else if (ret < 0) { isValid = false; continue; }
assert(b->core.l_qseq > 0);
qname = bam_get_canonical_name(b);
// if this is a paired-end read
if (ispaired == -1) ispaired = bam_is_paired(b);
else {
isValid = (ispaired == bam_is_paired(b));
if (!isValid) {
printf("\nWe detected both single-end and paired-end reads in the data!\nRSEM currently does not support a mixture of single-end/paired-end reads.\n");
continue;
}
}
if (ispaired) {
isValid = (sam_read1(in, header, b2) >= 0) && (qname == bam_get_canonical_name(b2)) && bam_is_paired(b2);
if (!isValid) {
printf("\nOnly find one mate for paired-end read %s!\nPlease make sure that the two mates of a paired-end read are adjacent to each other.\n", bam_get_qname(b));
continue;
}
assert(b2->core.l_qseq > 0);
isValid = (bam_is_read1(b) && bam_is_read2(b2)) || (bam_is_read1(b2) && bam_is_read2(b));
if (!isValid) {
printf("\nThe two mates of paired-end read %s are marked as both mate1 or both mate2!\n", bam_get_qname(b));
continue;
}
int value = int(bam_is_mapped(b)) + int(bam_is_mapped(b2));
isValid = (value != 1);
if (!isValid) {
printf("\nPaired-end read %s has an alignment with only one mate aligned!\n", bam_get_qname(b));
printf("Currently RSEM does not handle mixed alignments for paired-end reads.\n");
continue;
}
if (!bam_is_read1(b)) { bam1_t *tmp = b; b = b2; b2 = tmp; }
if (value == 2) {
isValid = (b->core.tid == b2->core.tid);
if (!isValid) {
printf("\nPaired-end read %s has a discordant alignment (two mates aligned to different reference sequences)!\n", bam_get_qname(b));
printf("Mate 1 aligns to %s and mate 2 aligns to %s\n", header->target_name[b->core.tid], header->target_name[b2->core.tid]);
printf("Currently RSEM does not handle discordant alignments.\n");
continue;
}
int strandedness = (int(bam_is_rev(b)) << 1) + int(bam_is_rev(b2));
isValid = (strandedness == 1 || strandedness == 2);
if (!isValid) {
printf("\nPaired-end read %s has an alignment in which two mates aligned to the same strand!\n", bam_get_qname(b));
printf("Its two mates aligned to %s in %s direction.\n", header->target_name[b->core.tid], (strandedness == 0 ? "forward" : "reverse"));
continue;
}
bam1_t *tb = (b->core.pos < b2->core.pos ? b : b2);
isValid = tb->core.pos >= 0 && tb->core.pos + abs(tb->core.isize) <= header->target_len[tb->core.tid];
if (!isValid) {
printf("\nPaired-end read %s aligns to [%d, %d) of transcript %s, which exceeds the transcript's boundary [0, %d)!\n",
bam_get_qname(b), tb->core.pos, tb->core.pos + abs(tb->core.isize), header->target_name[tb->core.tid], header->target_len[tb->core.tid]);
continue;
}
isValid = check_read(b, header);
if (!isValid) continue;
isValid = check_read(b2, header);
if (!isValid) continue;
}
readlen = b->core.l_qseq;
readlen2 = b2->core.l_qseq;
}
else {
if (bam_is_mapped(b)) {
isValid = check_read(b, header);
if (!isValid) continue;
}
readlen = b->core.l_qseq;
}
if (cqname != qname) {
isValid = used.find(qname) == used.end();
if (!isValid) { printf("\nThe alignments of read %s are not grouped together!\n", qname.c_str()); continue; }
used.insert(cqname);
cqname = qname;
creadlen = readlen;
if (ispaired) creadlen2 = readlen2;
}
else {
assert(cqname != "");
isValid = (creadlen == readlen && (!ispaired || creadlen2 == readlen2));
if (!isValid) { printf("\nRead %s have alignments showing different read/mate lengths!\n", qname.c_str()); continue; }
}
++cnt;
if (cnt % 1000000 == 0) { printf("."); fflush(stdout); }
} while(isValid);
bam_destroy1(b); bam_destroy1(b2);
bam_hdr_destroy(header);
sam_close(in);
if (isValid) printf("\nThe input file is valid!\n");
else printf("The input file is not valid!\n");
return 0;
}
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