picard_cpp/src/sam/markdups/markdups.cpp

/*
Description:
标记bam文件中的冗余信息，只处理按照坐标排序后的bam，且bam为单一样本数据

Copyright : All right reserved by ICT

Author : Zhang Zhonghai
Date : 2023/10/23
*/
#include <common/hts/bam_buf.h>
#include <common/utils/global_arg.h>
#include <common/utils/murmur3.h>
#include <common/utils/thpool.h>
#include <common/utils/timer.h>
#include <common/utils/util.h>
#include <common/utils/yarn.h>
#include <htslib/sam.h>
#include <htslib/thread_pool.h>
#include <sam/utils/read_ends.h>
#include <sam/utils/read_name_parser.h>

#include <iostream>
#include <queue>
#include <set>
#include <unordered_map>
#include <unordered_set>
#include <vector>
#include <string>

#include "dup_metrics.h"
#include "markdups_arg.h"
#include "md_funcs.h"
#include "parallel_md.h"
#include "serial_md.h"
#include "shared_args.h"

using namespace std;
using std::cout;
using std::string;

#define SMA_TAG_PG "PG"

#define BAM_BLOCK_SIZE 16L * 1024 * 1024
#define NO_SUCH_INDEX INT64_MAX

Timer tm_arr[20];  // 用来测试性能
/* 全局本地变量 */
vector<ReadNameParser> g_vRnParser;  // 每个线程一个read name parser
samFile *g_inBamFp;                  // 输入的bam文件
sam_hdr_t *g_inBamHeader;            // 输入的bam文件头信息
samFile *g_outBamFp = nullptr;       // 输出文件, sam或者bam格式
sam_hdr_t *g_outBamHeader;           // 输出文件的header

/* 参数对象作为全局对象，免得多次作为参数传入函数中 */
GlobalArg &g_gArg = GlobalArg::Instance();
static MarkDupsArg g_mdArg_;
MarkDupsArg &g_mdArg = g_mdArg_;
static GlobalDataArg gData_;
GlobalDataArg &gData = gData_;
DuplicationMetrics gMetrics_;
DuplicationMetrics &gMetrics = gMetrics_; // 记录统计信息

/* 程序版本等信息 */
const char *PROGRAM_GROUP_VERSION = "3.2.0";

// 调试信息
int zzhtestnum = 0;
int64_t bam_num1=0, bam_num2=0;
/**
 * Estimates the size of a library based on the number of paired end molecules observed
 * and the number of unique pairs observed.
 * <p>
 * Based on the Lander-Waterman equation that states:
 * C/X = 1 - exp( -N/X )
 * where
 * X = number of distinct molecules in library
 * N = number of read pairs
 * C = number of distinct fragments observed in read pairs
 */
static int64_t estimateLibrarySize(int64_t readPairs, int64_t uniqueReadPairs) {
    int64_t librarySize = 0;
    int64_t readPairDuplicates = readPairs - uniqueReadPairs;
    auto f = [](double x, double c, double n) { return c / x - 1 + exp(-n / x); };
    if (readPairs > 0 && readPairDuplicates > 0) {
        double m = 1.0;
        double M = 100.0;
        if (uniqueReadPairs >= readPairs || f(m * uniqueReadPairs, uniqueReadPairs, readPairs) < 0) {
            cerr << "Invalid values for pairs and unique pairs: " << readPairs << ", " << uniqueReadPairs << endl;
            return 0;
        }
        // find value of M, large enough to act as other side for bisection method
        while (f(M * uniqueReadPairs, uniqueReadPairs, readPairs) > 0) {
            M *= 10.0;
        }
        // use bisection method (no more than 40 times) to find solution
        for (int i = 0; i < 40; ++i) {
            double r = (m + M) / 2.0;
            double u = f(r * uniqueReadPairs, uniqueReadPairs, readPairs);
            if (u == 0)
                break;
            else if (u > 0)
                m = r;
            else if (u < 0)
                M = r;
        }
        return uniqueReadPairs * (m + M) / 2.0;
    }
    return librarySize;
}

/*
 * 获取文件名后缀
 */
string getFileExtension(const string &filename) {
    auto last_dot = filename.find_last_of('.');
    if (last_dot == string::npos) {
        return "";
    }
    return filename.substr(last_dot + 1);
}

/*
 * mark duplicate
 * 入口，假定bam是按照比对后的坐标排序的，同一个样本的话不需要考虑barcode的问题
 */
int MarkDuplicates(int argc, char *argv[]) {
    Timer::log_time("程序开始");
    Timer time_all;

    /* 读取命令行参数 */
    g_mdArg.parseArgument(argc, argv, &g_gArg);  // 解析命令行参数
    if (g_gArg.num_threads < 1)
        g_gArg.num_threads = 1;  // 线程数不能小于1

    /* 初始化一些参数和变量*/
    g_vRnParser.resize(g_gArg.num_threads);
    for (auto &parser : g_vRnParser)
        parser.SetReadNameRegex(g_mdArg.READ_NAME_REGEX);  // 用来解析read name中的tile，x，y信息

    /* 打开输入bam文件 */
    g_inBamFp = sam_open_format(g_gArg.in_fn.c_str(), "r", nullptr);
    if (!g_inBamFp) {
        Error("[%s] load sam/bam file failed.\n", __func__);
        return -1;
    }
    hts_set_opt(g_inBamFp, HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE);
    g_inBamHeader = sam_hdr_read(g_inBamFp);  // 读取header
    // 获取样本名称(libraryId)
    gMetrics.LIBRARY = sam_hdr_line_name(g_inBamHeader, "RG", 0);

    /* 利用线程池对输入输出文件进行读写 */
    htsThreadPool htsPoolRead = {NULL, 0};   // 多线程读取，创建线程池
    htsThreadPool htsPoolWrite = {NULL, 0};  // 读写用不同的线程池
    // htsPoolRead.pool = hts_tpool_init(g_gArg.num_threads);
    // htsPoolWrite.pool = hts_tpool_init(g_gArg.num_threads);
    htsPoolRead.pool = hts_tpool_init(32);
    htsPoolWrite.pool = hts_tpool_init(32);
    if (!htsPoolRead.pool || !htsPoolWrite.pool) {
        Error("[%d] failed to set up thread pool", __LINE__);
        sam_close(g_inBamFp);
        return -1;
    }
    hts_set_opt(g_inBamFp, HTS_OPT_THREAD_POOL, &htsPoolRead);

    /* 初始化输出文件 */
    char modeout[12] = "wb";
    sam_open_mode(modeout + 1, g_gArg.out_fn.c_str(), NULL);
    g_outBamFp = sam_open(g_gArg.out_fn.c_str(), modeout);
    g_outBamHeader = sam_hdr_dup(g_inBamHeader);
    // 修改输出文件的header
    sam_hdr_add_line(g_outBamHeader, "PG", "ID", g_mdArg.PROGRAM_RECORD_ID.c_str(), "VN", PROGRAM_GROUP_VERSION, "CL", 
                     (g_mdArg.PROGRAM_RECORD_ID + " " + g_gArg.GetArgValueString() + " " + g_mdArg.GetArgValueString()).c_str(), NULL);

    // 用同样的线程池处理输出文件
    hts_set_opt(g_outBamFp, HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE);
    hts_set_opt(g_outBamFp, HTS_OPT_THREAD_POOL, &htsPoolWrite);

    /* 冗余检查和标记 */
    if (g_gArg.num_threads == 1) {
        serialMarkDups();  // 串行运行
    } else {
        parallelMarkDups();  // 并行运行
    }

    /* 标记冗余, 将处理后的结果写入文件 */
    sam_close(g_inBamFp);  // 重新打开bam文件
    g_inBamFp = sam_open_format(g_gArg.in_fn.c_str(), "r", nullptr);
    if (!g_inBamFp) {
        Error("[%s] load sam/bam file failed.\n", __func__);
        return -1;
    }
    hts_set_opt(g_inBamFp, HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE);
    hts_set_opt(g_inBamFp, HTS_OPT_THREAD_POOL, &htsPoolRead);
    g_inBamHeader = sam_hdr_read(g_inBamFp);  // 读取header
    if (sam_hdr_write(g_outBamFp, g_outBamHeader) != 0) {
        Error("failed writing header to \"%s\"", g_gArg.out_fn.c_str());
        sam_close(g_outBamFp);
        sam_close(g_inBamFp);
        return -1;
    }
    // 输出index文件
    // string indexFn = g_gArg.out_fn + ".csi";  // 现在索引都是csi格式的
    string indexFn = g_gArg.out_fn + ".bai";  // min_shift = 0 是bai格式
    if ("sam" == getFileExtension(g_gArg.out_fn)) {
        indexFn = "";
    }

    if (!indexFn.empty()) {
        int index_min_shift = 0;
        if (g_mdArg.INDEX_FORMAT == ns_md::IndexFormat::CSI) {
            indexFn = g_gArg.out_fn + ".csi";
            index_min_shift = 14;
        }
        if (sam_idx_init(g_outBamFp, g_outBamHeader, 0 /*csi 14*/, indexFn.c_str()) < 0) {
            Error("failed to open index \"%s\" for writing", indexFn.c_str());
            sam_close(g_outBamFp);
            sam_close(g_inBamFp);
            return -1;
        }
    }
    // 读取输入文件
    // BamBufType inBuf(false);
    BamBufType inBuf(g_gArg.use_asyncio);
    inBuf.Init(g_inBamFp, g_inBamHeader, g_gArg.max_mem);
    DupIdxQueue<DupInfo> dupIdxQue, repIdxQue;
    DupIdxQueue<int64_t> opticalIdxQue;
    dupIdxQue.Init(&gData.dupIdxArr);
    repIdxQue.Init(&gData.repIdxArr);
    opticalIdxQue.Init(&gData.opticalDupIdxArr);
    Timer tw;
    cout << "dupsize: " << dupIdxQue.Size() << endl;
    uint64_t bamIdx = 0;
    DupInfo dupIdx = dupIdxQue.Pop();
    DupInfo repIdx = repIdxQue.Pop();
    uint64_t opticalIdx = opticalIdxQue.Pop();
    ByteBuf bytes;
    bam1_t *bp = bam_init1();
    bool isDup = false;
    bool isOpticalDup = false;
    bool isInDuplicateSet = false;
    uint32_t representativeReadIndexInFile = 0;
    uint32_t duplicateSetSize = 0;
    // exit(0);
    while (inBuf.ReadStat() >= 0) {
        Timer tw1;
        size_t readNum = inBuf.ReadBam();
        //    cout << "read: " << readNum << endl;
        for (size_t i = 0; i < inBuf.Size(); ++i) {
            BamWrap *bw = inBuf[i];
            if (bam_copy1(bp, bw->b) == nullptr) {
                Error("Can not copy sam record!");
                return -1;
            }
            bw->b = bp;
            isDup = false;
            isOpticalDup = false;
            isInDuplicateSet = false;
            // 删除原来的duplicate tag
            if (g_mdArg.CLEAR_DT) {
                uint8_t *oldTagVal = bam_aux_get(bw->b, g_mdArg.DUPLICATE_TYPE_TAG.c_str());
                if (oldTagVal != NULL) bam_aux_del(bw->b, oldTagVal);
            }
            ++bam_num2;
            // 统计信息
            if (bw->GetReadUnmappedFlag()) {
                ++gMetrics.UNMAPPED_READS;
            } else if (bw->IsSecondaryOrSupplementary()) {
                ++gMetrics.SECONDARY_OR_SUPPLEMENTARY_RDS;
            } else if (!bw->GetReadPairedFlag() || bw->GetMateUnmappedFlag()) {
                ++gMetrics.UNPAIRED_READS_EXAMINED;
            } else {
                ++gMetrics.READ_PAIRS_EXAMINED;  // will need to be divided by 2 at the end
            }

            /* 判断是否冗余 */
            if (bamIdx == dupIdx) {
                isDup = true;

                if (g_mdArg.TAG_DUPLICATE_SET_MEMBERS && dupIdx.dupSet != 0) {
                    // cerr << bamIdx << " " << dupIdx.repIdx << " " << dupIdx.dupSet << endl;
                    isInDuplicateSet = true;
                    representativeReadIndexInFile = dupIdx.repIdx;
                    duplicateSetSize = dupIdx.dupSet;
                }
                //  为了防止小内存运行的时候，有重复的dupidx，这时候dup的repIdx和dupSetSize可能会有不同
                while ((dupIdx = dupIdxQue.Pop()) == bamIdx);
                if (opticalIdx == bamIdx)
                    isOpticalDup = true;
                else if (opticalIdx < bamIdx) {
                    while ((opticalIdx = opticalIdxQue.Pop()) < bamIdx);
                    if (opticalIdx == bamIdx)
                        isOpticalDup = true;
                }
                // 添加冗余标识
                bw->SetDuplicateReadFlag(true);
                // if (isOpticalDup)
                //     cout << bamIdx << " optical" << endl;
                // else
                //     cout << bamIdx << " not opt" << endl;

                uint8_t *oldTagVal = bam_aux_get(bw->b, g_mdArg.DUPLICATE_TYPE_TAG.c_str());
                if (oldTagVal != NULL) bam_aux_del(bw->b, oldTagVal);
                if (isOpticalDup)
                    bam_aux_append(bw->b, g_mdArg.DUPLICATE_TYPE_TAG.c_str(), 'Z',
                                   g_mdArg.DUPLICATE_TYPE_SEQUENCING.size() + 1,
                                   (const uint8_t *)g_mdArg.DUPLICATE_TYPE_SEQUENCING.c_str());
                else
                    bam_aux_append(bw->b, g_mdArg.DUPLICATE_TYPE_TAG.c_str(), 'Z',
                                   g_mdArg.DUPLICATE_TYPE_LIBRARY.size() + 1,
                                   (const uint8_t *)g_mdArg.DUPLICATE_TYPE_LIBRARY.c_str());

                // cout << bamIdx << endl;
                // int ival = 2;
                // cout << "bam rec size: " << bw->b->l_data << "\t" << bw->b->m_data << endl;
                // if (bam_aux_append(bw->b, "DT", 'Z', 3, (const uint8_t*)"LB") < 0) Error("add tag error!");
                // // if (bam_aux_append(bw->b, "DT", 'i', sizeof(ival), (uint8_t *)&ival) < 0) Error("add tag error!");
                // cout << "bam rec size: " << bw->b->l_data << "\t" << bw->b->m_data << endl;

                // 计算统计信息
                if (!bw->IsSecondaryOrSupplementary() && !bw->GetReadUnmappedFlag()) {
                    // Update the duplication metrics
                    if (!bw->GetReadPairedFlag() || bw->GetMateUnmappedFlag()) {
                        ++gMetrics.UNPAIRED_READ_DUPLICATES;
                    } else {
                        ++gMetrics.READ_PAIR_DUPLICATES;  // will need to be divided by 2 at the end
                    }
                }
            } else {
                bw->SetDuplicateReadFlag(false);
            }
            if (g_mdArg.TAG_DUPLICATE_SET_MEMBERS && bamIdx == repIdx) {  // repressent
                // cerr << bamIdx << " " << repIdx.repIdx << " " << repIdx.dupSet << endl;
                isInDuplicateSet = true;
                representativeReadIndexInFile = repIdx.repIdx;
                duplicateSetSize = repIdx.dupSet;
                while (repIdx == bamIdx || repIdx.dupSet == 0) {
                    if (repIdxQue.Size() > 0)
                        repIdx = repIdxQue.Pop();
                    else {
                        repIdx = -1;
                        break;
                    }
                }
            }

            if (g_mdArg.TAG_DUPLICATE_SET_MEMBERS && isInDuplicateSet) {
                // cerr << bamIdx << " " << representativeReadIndexInFile << " " << duplicateSetSize << endl;
                uint8_t *oldTagVal = bam_aux_get(bw->b, g_mdArg.DUPLICATE_SET_INDEX_TAG.c_str());
                if (oldTagVal != NULL) bam_aux_del(bw->b, oldTagVal);
                bam_aux_append(bw->b, g_mdArg.DUPLICATE_SET_INDEX_TAG.c_str(), 'i',
                               sizeof(representativeReadIndexInFile), (uint8_t *)&representativeReadIndexInFile);
                oldTagVal = bam_aux_get(bw->b, g_mdArg.DUPLICATE_SET_SIZE_TAG.c_str());
                if (oldTagVal != NULL) bam_aux_del(bw->b, oldTagVal);
                bam_aux_append(bw->b, g_mdArg.DUPLICATE_SET_SIZE_TAG.c_str(), 'i', sizeof(duplicateSetSize),
                               (const uint8_t *)&duplicateSetSize);
            }
            // 每个read都要写到output，只是添加标识
            // cout << "bam rec size: " << bw->b->l_data << "\t" << bw->b->m_data << endl;
            // if (bamIdx == 922) break;
            ++bamIdx;
            if (isDup && g_mdArg.REMOVE_DUPLICATES) {
                continue;
            }
            if (isOpticalDup && g_mdArg.REMOVE_SEQUENCING_DUPLICATES) {
                continue;
            }
            if (!g_mdArg.PROGRAM_RECORD_ID.empty() && g_mdArg.ADD_PG_TAG_TO_READS) {
                uint8_t *oldTagVal = bam_aux_get(bw->b, "PG");
                if (oldTagVal != NULL) bam_aux_del(bw->b, oldTagVal);
                bam_aux_append(bw->b, "PG", 'Z', g_mdArg.PROGRAM_RECORD_ID.size() + 1,
                               (const uint8_t *)g_mdArg.PROGRAM_RECORD_ID.c_str());
            }
            if (sam_write1(g_outBamFp, g_outBamHeader, bw->b) < 0) {
                Error("failed writing sam record to \"%s\"", g_gArg.out_fn.c_str());
                sam_close(g_outBamFp);
                sam_close(g_inBamFp);
                return -1;
            }
        }
        inBuf.ClearAll();
        cout << "write round time: " << tw1.seconds_elapsed() << " s" << endl;
    }
    bam_destroy1(bp);
    cout << "bam_Num: " << bam_num1 << " : " << bam_num2 << endl;

    // 计算统计信息
    gMetrics.READ_PAIRS_EXAMINED /= 2;
    gMetrics.READ_PAIR_DUPLICATES /= 2;
    for (auto &arr : gData.opticalDupIdxArr) gMetrics.READ_PAIR_OPTICAL_DUPLICATES += arr.size();
    gMetrics.READ_PAIR_OPTICAL_DUPLICATES = gMetrics.READ_PAIR_OPTICAL_DUPLICATES / 2;
    gMetrics.ESTIMATED_LIBRARY_SIZE =
        estimateLibrarySize(gMetrics.READ_PAIRS_EXAMINED - gMetrics.READ_PAIR_OPTICAL_DUPLICATES,
                            gMetrics.READ_PAIRS_EXAMINED - gMetrics.READ_PAIR_DUPLICATES);
    if (gMetrics.UNPAIRED_READS_EXAMINED + gMetrics.READ_PAIRS_EXAMINED != 0) {
        gMetrics.PERCENT_DUPLICATION = (gMetrics.UNPAIRED_READ_DUPLICATES + gMetrics.READ_PAIR_DUPLICATES * 2) /
                                       (double)(gMetrics.UNPAIRED_READS_EXAMINED + gMetrics.READ_PAIRS_EXAMINED * 2);
    } else {
        gMetrics.PERCENT_DUPLICATION = 0;
    }

    cout << "metrics: \n"
         << "LIBRARY:                        " << gMetrics.LIBRARY << "\n"
         << "UNPAIRED_READS_EXAMINED:        " << gMetrics.UNPAIRED_READS_EXAMINED << "\n"
         << "READ_PAIRS_EXAMINED:            " << gMetrics.READ_PAIRS_EXAMINED << "\n"
         << "SECONDARY_OR_SUPPLEMENTARY_RDS: " << gMetrics.SECONDARY_OR_SUPPLEMENTARY_RDS << "\n"
         << "UNMAPPED_READS:                 " << gMetrics.UNMAPPED_READS << "\n"
         << "UNPAIRED_READ_DUPLICATES:       " << gMetrics.UNPAIRED_READ_DUPLICATES << "\n"
         << "READ_PAIR_DUPLICATES:           " << gMetrics.READ_PAIR_DUPLICATES << "\n"
         << "READ_PAIR_OPTICAL_DUPLICATES:   " << gMetrics.READ_PAIR_OPTICAL_DUPLICATES << "\n"
         << "PERCENT_DUPLICATION:            " << gMetrics.PERCENT_DUPLICATION << "\n"
         << "ESTIMATED_LIBRARY_SIZE:         " << gMetrics.ESTIMATED_LIBRARY_SIZE << endl;

    cout << "singleton pos: " << gMetrics.singletonReads.size() << endl;
    for (auto &itr : gMetrics.DuplicateCountHist) cout << "dup counts: " << itr.first << ": " << itr.second << endl;
    for (auto &itr : gMetrics.NonOpticalDuplicateCountHist)
        cout << "non optical dup counts: " << itr.first << ": " << itr.second << endl;
    for (auto &itr : gMetrics.OpticalDuplicatesCountHist)
        cout << "optical dup counts: " << itr.first << ": " << itr.second << endl;
    cout << "dupsize: " << dupIdxQue.Size() << "\t" << opticalIdxQue.Size() << "\t" << repIdxQue.Size() << endl;
    if (!indexFn.empty() && sam_idx_save(g_outBamFp) < 0) {
        Error("writing index failed");
        sam_close(g_outBamFp);
        sam_close(g_inBamFp);
        return -1;
    }
    cout << "write time: " << tw.seconds_elapsed() << " s" << endl;
    /* 关闭文件，收尾清理 */
    sam_close(g_outBamFp);
    sam_close(g_inBamFp);

    cout << "      总时间: " << time_all.seconds_elapsed() << endl;
    //    cout << "计算read end: " << tm_arr[0].acc_seconds_elapsed() << endl;
    Timer::log_time("程序结束");
    return 0;
}