From b97a8762fa9ccba1f046e0c68128a8166f8d4f85 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:45:28 -0800 Subject: [PATCH 01/22] src/original_duplicates.cpp: moving code to initialize original duplicates analysis here --- src/original_duplicates.cpp | 88 ++++++++++++++++++++++++++++++++++--- 1 file changed, 82 insertions(+), 6 deletions(-) diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index d0b960b..6fd00e8 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -2,10 +2,8 @@ #include "original_duplicates.hpp" -#include "bam_file.hpp" #include "duplication_results.hpp" #include "falco_file_format.hpp" -#include "fastq_bgzf_file.hpp" #include "file_info.hpp" #include "boost/boost_unordered.hpp" @@ -21,10 +19,88 @@ #include [[nodiscard]] auto -initialize_original_duplicates( - const std::vector &infiles, - const std::vector &infos, - const std::uint32_t n_threads) -> std::vector { +init_dups(const std::string &filename, const std::uint64_t n_unique) + -> dups_map_t { + static constexpr auto complem = [](const auto x) { + return "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; + }; + static constexpr auto n_unique_multiplier = 10; + const std::unique_ptr in( + hts_open(std::data(filename), "r"), &hts_close); + if (!in) + throw std::system_error(std::make_error_code(std::errc(errno)), + "failed to open file: " + filename); + const std::unique_ptr h( + sam_hdr_read(in.get()), &sam_hdr_destroy); + if (!h) + throw std::system_error(std::make_error_code(std::errc(errno)), + "failed to read header: " + filename); + // NOLINTNEXTLINE(cppcoreguidelines-pro-type-union-access) + std::unique_ptr rec(bam_init1(), &bam_destroy1); + + dups_map_t dups; + std::vector buffer; + int r{}; + std::uint64_t n_reads{}; + const auto max_n_reads = n_unique_multiplier * n_unique; + while (n_reads++ < max_n_reads && std::size(dups) < n_unique && + (r = sam_read1(in.get(), h.get(), rec.get())) >= 0) { + const auto l_qseq = rec->core.l_qseq; + const auto seq = bam_get_seq(rec.get()); + if (std::ssize(buffer) < l_qseq) + buffer.resize(l_qseq); + for (auto i = 0; i < l_qseq; ++i) + buffer[i] = seq_nt16_str[bam_seqi(seq, i)]; + if (bam_is_rev(rec.get())) { + std::reverse(std::begin(buffer), std::begin(buffer) + l_qseq); + std::transform(std::cbegin(buffer), std::cbegin(buffer) + l_qseq, + std::begin(buffer), complem); + } + ++dups[falco_word(std::data(buffer), l_qseq)]; + } + if (r < -1) // error + throw std::system_error(std::make_error_code(std::errc(errno)), + "error reading bam record from: " + filename); + return dups; +} + +[[nodiscard]] auto +init_dups_fq(const std::string &filename, const std::uint64_t n_unique) + -> dups_map_t { + static constexpr auto n_unique_multiplier = 10; + const std::unique_ptr in( + bgzf_open(std::data(filename), "r"), &bgzf_close); + if (!in) + throw std::system_error(std::make_error_code(std::errc(errno)), + "failed to open file: " + filename); + dups_map_t dups; + kstring_t s = KS_INITIALIZE; + int r{}; + std::uint64_t n_lines{}; + std::uint64_t n_reads{}; + const auto max_n_reads = n_unique_multiplier * n_unique; + while (n_reads < max_n_reads && std::size(dups) < n_unique && + (r = bgzf_getline(in.get(), '\n', &s)) >= 0) { + if (n_lines % 4 == 1) { + const auto l_qseq = ks_len(&s); + const auto seq = ks_c_str(&s); + ++dups[falco_word(seq, l_qseq)]; + ++n_reads; + } + ++n_lines; + } + if (r < -1) // error + throw std::system_error(std::make_error_code(std::errc(errno)), + "error reading bam record from: " + filename); + ks_free(&s); + return dups; +} + +[[nodiscard]] auto +initialize_original_duplicates(const std::vector &infiles, + const std::vector &infos, + const std::uint32_t n_threads) + -> std::vector { assert(std::size(infiles) == std::size(infos)); const std::uint64_t n_files = std::size(infiles); std::vector dups(n_files); From 0e0bfc3c5c9afa1147b0e5d4cef298d55df0f38f Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:45:49 -0800 Subject: [PATCH 02/22] src/fastq_bgzf_file.hcpp and src/bam_file.hcpp: moving code for initializing original duplication data structures to original_duplicates.cpp --- src/bam_file.cpp | 46 ----------------------------------------- src/bam_file.hpp | 4 ---- src/fastq_bgzf_file.cpp | 32 ---------------------------- src/fastq_bgzf_file.hpp | 4 ---- 4 files changed, 86 deletions(-) diff --git a/src/bam_file.cpp b/src/bam_file.cpp index 90533a8..d160a56 100644 --- a/src/bam_file.cpp +++ b/src/bam_file.cpp @@ -65,52 +65,6 @@ estimate_n_reads_bam(const std::string &filename) return {n_reads_estimate, read_len_estimate, filesize}; } -[[nodiscard]] auto -init_dups(const std::string &filename, const std::uint64_t n_unique) - -> dups_map_t { - static constexpr auto complem = [](const auto x) { - return "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; - }; - static constexpr auto n_unique_multiplier = 10; - const std::unique_ptr in( - hts_open(std::data(filename), "r"), &hts_close); - if (!in) - throw std::system_error(std::make_error_code(std::errc(errno)), - "failed to open file: " + filename); - const std::unique_ptr h( - sam_hdr_read(in.get()), &sam_hdr_destroy); - if (!h) - throw std::system_error(std::make_error_code(std::errc(errno)), - "failed to read header: " + filename); - // NOLINTNEXTLINE(cppcoreguidelines-pro-type-union-access) - std::unique_ptr rec(bam_init1(), &bam_destroy1); - - dups_map_t dups; - std::vector buffer; - int r{}; - std::uint64_t n_reads{}; - const auto max_n_reads = n_unique_multiplier * n_unique; - while (n_reads++ < max_n_reads && std::size(dups) < n_unique && - (r = sam_read1(in.get(), h.get(), rec.get())) >= 0) { - const auto l_qseq = rec->core.l_qseq; - const auto seq = bam_get_seq(rec.get()); - if (std::ssize(buffer) < l_qseq) - buffer.resize(l_qseq); - for (auto i = 0; i < l_qseq; ++i) - buffer[i] = seq_nt16_str[bam_seqi(seq, i)]; - if (bam_is_rev(rec.get())) { - std::reverse(std::begin(buffer), std::begin(buffer) + l_qseq); - std::transform(std::cbegin(buffer), std::cbegin(buffer) + l_qseq, - std::begin(buffer), complem); - } - ++dups[falco_word(std::data(buffer), l_qseq)]; - } - if (r < -1) // error - throw std::system_error(std::make_error_code(std::errc(errno)), - "error reading bam record from: " + filename); - return dups; -} - auto bam_file::load_next(const std::int32_t file_id, task_queue &tq, diff --git a/src/bam_file.hpp b/src/bam_file.hpp index 97a2473..10c5458 100644 --- a/src/bam_file.hpp +++ b/src/bam_file.hpp @@ -129,10 +129,6 @@ class bam_file { estimate_n_reads_bam(const std::string &filename) -> std::tuple; -[[nodiscard]] auto -init_dups(const std::string &filename, const std::uint64_t n_unique) - -> dups_map_t; - inline auto make_tasks(bam_file &reads_file, const std::int64_t n_threads, diff --git a/src/fastq_bgzf_file.cpp b/src/fastq_bgzf_file.cpp index 719e928..0c3605c 100644 --- a/src/fastq_bgzf_file.cpp +++ b/src/fastq_bgzf_file.cpp @@ -55,38 +55,6 @@ estimate_n_reads_fastq_bgzf(const std::string &filename) return {static_cast(n_reads_est), read_len_est, filesize}; } -[[nodiscard]] auto -init_dups_fq(const std::string &filename, const std::uint64_t n_unique) - -> dups_map_t { - static constexpr auto n_unique_multiplier = 10; - const std::unique_ptr in( - bgzf_open(std::data(filename), "r"), &bgzf_close); - if (!in) - throw std::system_error(std::make_error_code(std::errc(errno)), - "failed to open file: " + filename); - dups_map_t dups; - kstring_t s = KS_INITIALIZE; - int r{}; - std::uint64_t n_lines{}; - std::uint64_t n_reads{}; - const auto max_n_reads = n_unique_multiplier * n_unique; - while (n_reads < max_n_reads && std::size(dups) < n_unique && - (r = bgzf_getline(in.get(), '\n', &s)) >= 0) { - if (n_lines % 4 == 1) { - const auto l_qseq = ks_len(&s); - const auto seq = ks_c_str(&s); - ++dups[falco_word(seq, l_qseq)]; - ++n_reads; - } - ++n_lines; - } - if (r < -1) // error - throw std::system_error(std::make_error_code(std::errc(errno)), - "error reading bam record from: " + filename); - ks_free(&s); - return dups; -} - auto fastq_bgzf_file::load_next(const std::int32_t file_id, task_queue &tq, diff --git a/src/fastq_bgzf_file.hpp b/src/fastq_bgzf_file.hpp index 0023291..26a2169 100644 --- a/src/fastq_bgzf_file.hpp +++ b/src/fastq_bgzf_file.hpp @@ -128,10 +128,6 @@ class fastq_bgzf_file { estimate_n_reads_fastq_bgzf(const std::string &filename) -> std::tuple; -[[nodiscard]] auto -init_dups_fq(const std::string &filename, const std::uint64_t n_unique) - -> dups_map_t; - inline auto make_tasks(fastq_bgzf_file &reads_file, const std::int64_t n_threads, From 754dfd79119f7d34ca14df51806001b81a0953b6 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:47:34 -0800 Subject: [PATCH 03/22] src/original_duplicates.cpp: adding forgotten headers --- src/original_duplicates.cpp | 16 ++++++++++------ 1 file changed, 10 insertions(+), 6 deletions(-) diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index 6fd00e8..7f369a3 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -8,6 +8,10 @@ #include "boost/boost_unordered.hpp" +#include // IWYU pragma: keep +#include +#include + #include #include #include @@ -19,7 +23,7 @@ #include [[nodiscard]] auto -init_dups(const std::string &filename, const std::uint64_t n_unique) +init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { static constexpr auto complem = [](const auto x) { return "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; @@ -65,7 +69,7 @@ init_dups(const std::string &filename, const std::uint64_t n_unique) } [[nodiscard]] auto -init_dups_fq(const std::string &filename, const std::uint64_t n_unique) +init_orig_dups_fq(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { static constexpr auto n_unique_multiplier = 10; const std::unique_ptr in( @@ -123,10 +127,10 @@ initialize_original_duplicates(const std::vector &infiles, } dups[file_id] = falco::is_mapped_reads(infos[file_id].format) - ? init_dups(infiles[file_id], - duplication_results::max_reads_to_hash) - : init_dups_fq(infiles[file_id], - duplication_results::max_reads_to_hash); + ? init_orig_dups_bam(infiles[file_id], + duplication_results::max_reads_to_hash) + : init_orig_dups_fq(infiles[file_id], + duplication_results::max_reads_to_hash); } }); std::ranges::for_each(workers, [](auto &w) { w.join(); }); From feccee05cb99a4317c1a8c4d62cd5466fcbf9bc4 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:55:14 -0800 Subject: [PATCH 04/22] src/original_duplicates.cpp: adding to_acgtn in both the init_orig_dups_bam and init_orig_dups_fq to convert all non-ACGT letters to N prior to forming a falco_word when initializing the unique 'reads' for original duplicates analysis --- src/original_duplicates.cpp | 26 ++++++++++++++++++++++---- 1 file changed, 22 insertions(+), 4 deletions(-) diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index 7f369a3..f25d54a 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -25,10 +25,19 @@ [[nodiscard]] auto init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { - static constexpr auto complem = [](const auto x) { - return "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; - }; + // ADS: n_unique_multiplier: contributes to max reads to examine, multiplying + // the n_unique that is the target; impossible to know how many needed to hit + // the target, so try to overshoot. static constexpr auto n_unique_multiplier = 10; + static constexpr auto complem = [](char &x) { + assert(x >= 'A' && x <= 'T'); + x = "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; + }; + static constexpr auto to_acgtn = [](const char x) { + static constexpr char mask = 15; + static constexpr std::span seq_nt16_str_acgtn = "NACNGNNNTNNNNNNN"; + return seq_nt16_str_acgtn[x & mask]; + }; const std::unique_ptr in( hts_open(std::data(filename), "r"), &hts_close); if (!in) @@ -54,7 +63,7 @@ init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) if (std::ssize(buffer) < l_qseq) buffer.resize(l_qseq); for (auto i = 0; i < l_qseq; ++i) - buffer[i] = seq_nt16_str[bam_seqi(seq, i)]; + buffer[i] = to_acgtn(bam_seqi(seq, i)); if (bam_is_rev(rec.get())) { std::reverse(std::begin(buffer), std::begin(buffer) + l_qseq); std::transform(std::cbegin(buffer), std::cbegin(buffer) + l_qseq, @@ -71,7 +80,15 @@ init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) [[nodiscard]] auto init_orig_dups_fq(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { + // ADS: n_unique_multiplier: contributes to max reads to examine, multiplying + // the n_unique that is the target; impossible to know how many needed to hit + // the target, so try to overshoot. static constexpr auto n_unique_multiplier = 10; + static constexpr auto to_acgtn = [](const auto c) { + static constexpr auto mask = 31; + static constexpr std::span to_acgtn_sp = "NANCNNNGNNNNNNNNNNNNTNNNNNNNNNNN"; + return to_acgtn_sp[c & mask]; + }; const std::unique_ptr in( bgzf_open(std::data(filename), "r"), &bgzf_close); if (!in) @@ -87,6 +104,7 @@ init_orig_dups_fq(const std::string &filename, const std::uint64_t n_unique) (r = bgzf_getline(in.get(), '\n', &s)) >= 0) { if (n_lines % 4 == 1) { const auto l_qseq = ks_len(&s); + std::ranges::transform(std::span(s.s, l_qseq), s.s, to_acgtn); const auto seq = ks_c_str(&s); ++dups[falco_word(seq, l_qseq)]; ++n_reads; From 03bcc802d59099e7cf25ace004f1258dc25cea58 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:56:38 -0800 Subject: [PATCH 05/22] CMakeLists.txt: original_duplicates object links to HTSLIB so that the htslib headers will be visibile when making original_duplicates.o --- CMakeLists.txt | 2 ++ 1 file changed, 2 insertions(+) diff --git a/CMakeLists.txt b/CMakeLists.txt index b4c0948..c7a7d41 100644 --- a/CMakeLists.txt +++ b/CMakeLists.txt @@ -130,6 +130,8 @@ target_link_libraries(falco_file_format PRIVATE HTSLIB::HTSLIB) target_link_libraries(tile_processor PRIVATE HTSLIB::HTSLIB) target_link_libraries(quality_score PRIVATE HTSLIB::HTSLIB) target_link_libraries(fastq_gz_file PRIVATE HTSLIB::HTSLIB) +target_link_libraries(original_duplicates PRIVATE HTSLIB::HTSLIB) + if(USE_ISAL) target_link_libraries(fastq_gz_file PUBLIC ISAL::ISAL) endif() From 91e83cbf26282c99c4cf41d25b15f62fd83e6b4d Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 17:00:44 -0800 Subject: [PATCH 06/22] src/original_duplicates.cpp: fix changing transform to for_each_n when complementing the already reversed read sequence for revcomp mappers --- src/original_duplicates.cpp | 3 +-- 1 file changed, 1 insertion(+), 2 deletions(-) diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index f25d54a..66c71da 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -66,8 +66,7 @@ init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) buffer[i] = to_acgtn(bam_seqi(seq, i)); if (bam_is_rev(rec.get())) { std::reverse(std::begin(buffer), std::begin(buffer) + l_qseq); - std::transform(std::cbegin(buffer), std::cbegin(buffer) + l_qseq, - std::begin(buffer), complem); + std::for_each_n(std::begin(buffer), l_qseq, complem); } ++dups[falco_word(std::data(buffer), l_qseq)]; } From 8a2a40e2a241b70175d7e5feb92547a1baabc240 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 17:43:16 -0800 Subject: [PATCH 07/22] static analysis --- src/bam_file.cpp | 2 -- src/bam_file.hpp | 1 - src/duplication_results.cpp | 3 +++ src/fastq_bgzf_file.cpp | 4 +--- src/fastq_bgzf_file.hpp | 1 - src/original_duplicates.cpp | 8 +++++++- 6 files changed, 11 insertions(+), 8 deletions(-) diff --git a/src/bam_file.cpp b/src/bam_file.cpp index d160a56..b8a28f7 100644 --- a/src/bam_file.cpp +++ b/src/bam_file.cpp @@ -2,9 +2,7 @@ #include "bam_file.hpp" #include "bamrec.hpp" -#include "duplication_results.hpp" #include "falco_utils.hpp" -#include "falco_word.hpp" #include "task_queue.hpp" #include // IWYU pragma: keep diff --git a/src/bam_file.hpp b/src/bam_file.hpp index 10c5458..f781c89 100644 --- a/src/bam_file.hpp +++ b/src/bam_file.hpp @@ -6,7 +6,6 @@ #include "bam_header.hpp" #include "bgzf_block.hpp" #include "bgzf_reader.hpp" -#include "duplication_results.hpp" #include #include diff --git a/src/duplication_results.cpp b/src/duplication_results.cpp index 0a33242..ff269dc 100644 --- a/src/duplication_results.cpp +++ b/src/duplication_results.cpp @@ -27,6 +27,7 @@ #include #include #include +#include #include #include #include @@ -236,6 +237,8 @@ duplication_results::get_dups_summary(const std::uint64_t n_reads) const std::vector hist_dedup(max_dup + 1); for (const auto n_copies : std::views::values(dups)) ++hist_dedup[n_copies]; + if (hist_dedup[0] > 0) + throw std::runtime_error("Non-zero count of indexed reads seen 0 times"); for (auto [idx, val] : falco::views::enumerate(hist_dedup)) val = static_cast( get_corrected_count(count_at_limit, n_reads, idx, val)); diff --git a/src/fastq_bgzf_file.cpp b/src/fastq_bgzf_file.cpp index 0c3605c..68536e5 100644 --- a/src/fastq_bgzf_file.cpp +++ b/src/fastq_bgzf_file.cpp @@ -1,15 +1,13 @@ // SPDX-License-Identifier: MIT; Copyright 2026 Andrew D Smith #include "fastq_bgzf_file.hpp" -#include "duplication_results.hpp" #include "falco_utils.hpp" -#include "falco_word.hpp" #include "fqrec.hpp" #include "task_queue.hpp" #include #include -#include +#include // IWYU pragma: keep #include #include diff --git a/src/fastq_bgzf_file.hpp b/src/fastq_bgzf_file.hpp index 26a2169..8656e2d 100644 --- a/src/fastq_bgzf_file.hpp +++ b/src/fastq_bgzf_file.hpp @@ -5,7 +5,6 @@ #include "bgzf_block.hpp" #include "bgzf_reader.hpp" -#include "duplication_results.hpp" #include #include diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index 66c71da..44629d5 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -4,24 +4,29 @@ #include "duplication_results.hpp" #include "falco_file_format.hpp" +#include "falco_word.hpp" #include "file_info.hpp" #include "boost/boost_unordered.hpp" #include // IWYU pragma: keep -#include #include #include #include +#include #include +#include #include #include +#include #include +#include #include #include // for std::move #include +namespace { [[nodiscard]] auto init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { @@ -116,6 +121,7 @@ init_orig_dups_fq(const std::string &filename, const std::uint64_t n_unique) ks_free(&s); return dups; } +} // namespace [[nodiscard]] auto initialize_original_duplicates(const std::vector &infiles, From a143908427c6aba941f2271b678fb20a80b9d235 Mon Sep 17 00:00:00 2001 From: Maximilian Stammnitz Date: Mon, 2 Nov 2026 14:49:55 +0100 Subject: [PATCH 08/22] src: grade adapter, overrepresented, N content and duplication as FastQC Four modules were graded against cutoffs in different units from the values compared with them, or on a different quantity from the one the report shows: - overrepresented: the value was a fraction of reads, the default cutoffs (0.1, 1) are percent, so the effective cutoffs were 10% and 100%. The grade is now the percentage of the top sequence in the reported list (zero if the list is empty), as in FastQC, so a sequence below the list's own cutoffs can no longer raise it. - adapter: the value was the largest per-position count of an adapter start, while the report (and FastQC) shows the cumulative percentage of reads with the adapter at or before each position. The grade is now the maximum of that cumulative percentage. - n_content, duplication: fractions compared with fractional default cutoffs, which worked, but a config file in the FastQC limits.txt format (percent: n_content 5/20, duplication 70/50) made N content unable to warn and duplication always fail. Both are now graded in percent and the defaults are given in percent, like every other module and like Configuration/limits.txt. Also, for modules where higher is worse, a value equal to a cutoff no longer reaches it (FastQC tests value > cutoff), e.g. a sequence at exactly 0.1% passes. data/test_data/md5sum.txt: bam_2 now warns for overrepresented sequences (top sequence 0.12% of reads), as FastQC 0.13.0 does. --- data/test_data/md5sum.txt | 2 +- src/adapter_matcher.cpp | 13 ++++++++----- src/duplication_results.cpp | 13 +++++++------ src/falco_grade.cpp | 14 ++++++++------ 4 files changed, 24 insertions(+), 18 deletions(-) diff --git a/data/test_data/md5sum.txt b/data/test_data/md5sum.txt index d227693..0987ae9 100644 --- a/data/test_data/md5sum.txt +++ b/data/test_data/md5sum.txt @@ -1,6 +1,6 @@ e4f4641559aadc9ec7b5c50cc4aa82e3 bam_kmers_out/bam_1/fastqc_data.txt 090a74739499d287a7aeea689a78b4e9 bam_mult_out/bam_1/fastqc_data.txt -7cc30a3a14796140f05940485ab1bed5 bam_mult_out/bam_2/fastqc_data.txt +e3568c45f8cc358c263e51c4ece215cd bam_mult_out/bam_2/fastqc_data.txt 090a74739499d287a7aeea689a78b4e9 bam_orig_dups_out/bam_1/fastqc_data.txt 090a74739499d287a7aeea689a78b4e9 bam_out/bam_1/fastqc_data.txt 090a74739499d287a7aeea689a78b4e9 bam_tiles_out/bam_1/fastqc_data.txt diff --git a/src/adapter_matcher.cpp b/src/adapter_matcher.cpp index bc9552c..e088e15 100644 --- a/src/adapter_matcher.cpp +++ b/src/adapter_matcher.cpp @@ -55,13 +55,16 @@ adapter_matcher::add_and_consume(adapter_matcher &&rhs) -> void { [[nodiscard]] auto adapter_matcher::get_grade(const std::uint64_t n_reads) const -> std::string { static constexpr auto label = "adapter"; - const auto max_count = - adap_counts.empty() - ? 0 - : std::ranges::max(adap_counts | std::views::transform(std::ranges::max)); + // The report shows, per adapter, the cumulative percentage of reads with the + // adapter starting at or before each position; as in FastQC, the grade is + // the maximum of that, i.e. the total count of each adapter over positions. + std::vector totals(n_adapters, 0); + for (const auto &counts : adap_counts) + std::ranges::transform(totals, counts, std::begin(totals), std::plus{}); + const auto max_count = totals.empty() ? 0 : std::ranges::max(totals); return grader_set::get_grade( label, - as_frac(max_count, std::max(static_cast(1), n_reads))); + pct(as_frac(max_count, std::max(static_cast(1), n_reads)))); } [[nodiscard]] auto diff --git a/src/duplication_results.cpp b/src/duplication_results.cpp index 0a33242..bfdb405 100644 --- a/src/duplication_results.cpp +++ b/src/duplication_results.cpp @@ -157,11 +157,12 @@ duplication_results::add_and_consume(duplication_results &rhs) -> void { get_grade_overrepresented(const std::uint64_t n_reads, const duplication_results &dr) -> std::string { static constexpr auto label = "overrepresented"; - const auto max_n_obs = - dr.dups.empty() ? 0LU : std::ranges::max(std::views::values(dr.dups)); - return grader_set::get_grade( - label, - as_frac(max_n_obs, std::max(static_cast(1), n_reads))); + // As in FastQC, grade on the list the report shows: the percentage of the + // most frequent listed sequence (in percent, like the cutoffs), or zero if + // no sequence passes the list's cutoffs. + const auto overrep = dr.get_overrepresented(n_reads); + return grader_set::get_grade(label, + overrep.empty() ? 0.0 : overrep.front().pct_val); } [[nodiscard]] auto @@ -283,7 +284,7 @@ get_grade_duplication(const dup_summary_t &summary) -> std::string { }; const auto frac_dedup = as_frac(reduce(summary.hist_dedup), reduce(summary.hist_mass)); - return grader_set::get_grade(label, frac_dedup); + return grader_set::get_grade(label, pct(frac_dedup)); } [[nodiscard]] auto diff --git a/src/falco_grade.cpp b/src/falco_grade.cpp index 28896e0..7c558e7 100644 --- a/src/falco_grade.cpp +++ b/src/falco_grade.cpp @@ -24,15 +24,15 @@ static const auto default_graders = std::array{ // NOLINT(cert-err58-cpp) grader{.name = "quality_base_median", .warn = 25.0, .fail = 20.0}, grader{.name = "quality_base_lower", .warn = 10.0, .fail = 5.00}, grader{.name = "quality_sequence", .warn = 27.0, .fail = 20.0}, - grader{.name = "n_content", .warn = 0.05, .fail = 0.20}, + grader{.name = "n_content", .warn = 5.00, .fail = 20.0}, grader{.name = "sequence", .warn = 10.0, .fail = 20.0}, grader{.name = "sequence_length", .warn = 1.00, .fail = 1.00}, grader{.name = "gc_sequence", .warn = 15.0, .fail = 30.0}, - grader{.name = "duplication", .warn = 0.70, .fail = 0.50}, + grader{.name = "duplication", .warn = 70.0, .fail = 50.0}, grader{.name = "overrepresented", .warn = 0.10, .fail = 1.00}, grader{.name = "kmer", .warn = 2.00, .fail = 5.00}, grader{.name = "tile", .warn = 5.00, .fail = 10.0}, - grader{.name = "adapter", .warn = 0.05, .fail = 0.10}, + grader{.name = "adapter", .warn = 5.00, .fail = 10.0}, }; // clang-format on @@ -74,9 +74,11 @@ file_grades::get_title(const std::string &name) -> std::string { grader::identify_grade(const double value) const -> std::string { using std::string_literals::operator""s; if (warn < fail) { - if (value < warn) + // higher is worse: as in FastQC, a value equal to a cutoff does not + // reach it (e.g., 0.1% overrepresented passes, more than 0.1% warns) + if (value <= warn) return "pass"s; - if (value < fail) + if (value <= fail) return "warn"s; return "fail"s; } @@ -198,7 +200,7 @@ get_grade_n_content(const std::vector &n_counts, std::reduce(std::cbegin(nucs[max_idx]), std::cend(nucs[max_idx])); const auto max_n_frac = as_frac(n_counts[max_idx], n_counts[max_idx] + total_non_n); - return grader_set::get_grade(label, max_n_frac); + return grader_set::get_grade(label, pct(max_n_frac)); } [[nodiscard]] auto From a2a29ed7f9128b2912334e604d21648a8b4244af Mon Sep 17 00:00:00 2001 From: Maximilian Stammnitz Date: Mon, 9 Nov 2026 13:45:09 +0100 Subject: [PATCH 09/22] src: added my initials/email to the comments blocks for the grading fix --- src/adapter_matcher.cpp | 1 + src/duplication_results.cpp | 1 + src/falco_grade.cpp | 1 + 3 files changed, 3 insertions(+) diff --git a/src/adapter_matcher.cpp b/src/adapter_matcher.cpp index e088e15..7646c80 100644 --- a/src/adapter_matcher.cpp +++ b/src/adapter_matcher.cpp @@ -55,6 +55,7 @@ adapter_matcher::add_and_consume(adapter_matcher &&rhs) -> void { [[nodiscard]] auto adapter_matcher::get_grade(const std::uint64_t n_reads) const -> std::string { static constexpr auto label = "adapter"; + // MRS (maximilian.stammnitz@crg.eu) with Claude Opus 5.5: // The report shows, per adapter, the cumulative percentage of reads with the // adapter starting at or before each position; as in FastQC, the grade is // the maximum of that, i.e. the total count of each adapter over positions. diff --git a/src/duplication_results.cpp b/src/duplication_results.cpp index bfdb405..ea558db 100644 --- a/src/duplication_results.cpp +++ b/src/duplication_results.cpp @@ -157,6 +157,7 @@ duplication_results::add_and_consume(duplication_results &rhs) -> void { get_grade_overrepresented(const std::uint64_t n_reads, const duplication_results &dr) -> std::string { static constexpr auto label = "overrepresented"; + // MRS (maximilian.stammnitz@crg.eu) with Claude Opus 5.5: // As in FastQC, grade on the list the report shows: the percentage of the // most frequent listed sequence (in percent, like the cutoffs), or zero if // no sequence passes the list's cutoffs. diff --git a/src/falco_grade.cpp b/src/falco_grade.cpp index 7c558e7..c8c40bc 100644 --- a/src/falco_grade.cpp +++ b/src/falco_grade.cpp @@ -74,6 +74,7 @@ file_grades::get_title(const std::string &name) -> std::string { grader::identify_grade(const double value) const -> std::string { using std::string_literals::operator""s; if (warn < fail) { + // MRS (maximilian.stammnitz@crg.eu) with Claude Opus 5.5: // higher is worse: as in FastQC, a value equal to a cutoff does not // reach it (e.g., 0.1% overrepresented passes, more than 0.1% warns) if (value <= warn) From e34c1872739b33b628aaf770bcebdf80d7e670a0 Mon Sep 17 00:00:00 2001 From: Maximilian Stammnitz Date: Mon, 9 Nov 2026 23:24:14 +0100 Subject: [PATCH 10/22] update comments --- src/adapter_matcher.cpp | 2 +- src/duplication_results.cpp | 2 +- src/falco_grade.cpp | 2 +- 3 files changed, 3 insertions(+), 3 deletions(-) diff --git a/src/adapter_matcher.cpp b/src/adapter_matcher.cpp index 7646c80..bc0ea1e 100644 --- a/src/adapter_matcher.cpp +++ b/src/adapter_matcher.cpp @@ -55,7 +55,7 @@ adapter_matcher::add_and_consume(adapter_matcher &&rhs) -> void { [[nodiscard]] auto adapter_matcher::get_grade(const std::uint64_t n_reads) const -> std::string { static constexpr auto label = "adapter"; - // MRS (maximilian.stammnitz@crg.eu) with Claude Opus 5.5: + // MRS (maximilian.stammnitz@crg.eu): // The report shows, per adapter, the cumulative percentage of reads with the // adapter starting at or before each position; as in FastQC, the grade is // the maximum of that, i.e. the total count of each adapter over positions. diff --git a/src/duplication_results.cpp b/src/duplication_results.cpp index ea558db..b5a3f0d 100644 --- a/src/duplication_results.cpp +++ b/src/duplication_results.cpp @@ -157,7 +157,7 @@ duplication_results::add_and_consume(duplication_results &rhs) -> void { get_grade_overrepresented(const std::uint64_t n_reads, const duplication_results &dr) -> std::string { static constexpr auto label = "overrepresented"; - // MRS (maximilian.stammnitz@crg.eu) with Claude Opus 5.5: + // MRS (maximilian.stammnitz@crg.eu): // As in FastQC, grade on the list the report shows: the percentage of the // most frequent listed sequence (in percent, like the cutoffs), or zero if // no sequence passes the list's cutoffs. diff --git a/src/falco_grade.cpp b/src/falco_grade.cpp index c8c40bc..d2133a9 100644 --- a/src/falco_grade.cpp +++ b/src/falco_grade.cpp @@ -74,7 +74,7 @@ file_grades::get_title(const std::string &name) -> std::string { grader::identify_grade(const double value) const -> std::string { using std::string_literals::operator""s; if (warn < fail) { - // MRS (maximilian.stammnitz@crg.eu) with Claude Opus 5.5: + // MRS (maximilian.stammnitz@crg.eu): // higher is worse: as in FastQC, a value equal to a cutoff does not // reach it (e.g., 0.1% overrepresented passes, more than 0.1% warns) if (value <= warn) From 9c12e9395fb96094badeae42b0d09f55ff1e590c Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:45:28 -0800 Subject: [PATCH 11/22] src/original_duplicates.cpp: moving code to initialize original duplicates analysis here --- src/original_duplicates.cpp | 88 ++++++++++++++++++++++++++++++++++--- 1 file changed, 82 insertions(+), 6 deletions(-) diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index d0b960b..6fd00e8 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -2,10 +2,8 @@ #include "original_duplicates.hpp" -#include "bam_file.hpp" #include "duplication_results.hpp" #include "falco_file_format.hpp" -#include "fastq_bgzf_file.hpp" #include "file_info.hpp" #include "boost/boost_unordered.hpp" @@ -21,10 +19,88 @@ #include [[nodiscard]] auto -initialize_original_duplicates( - const std::vector &infiles, - const std::vector &infos, - const std::uint32_t n_threads) -> std::vector { +init_dups(const std::string &filename, const std::uint64_t n_unique) + -> dups_map_t { + static constexpr auto complem = [](const auto x) { + return "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; + }; + static constexpr auto n_unique_multiplier = 10; + const std::unique_ptr in( + hts_open(std::data(filename), "r"), &hts_close); + if (!in) + throw std::system_error(std::make_error_code(std::errc(errno)), + "failed to open file: " + filename); + const std::unique_ptr h( + sam_hdr_read(in.get()), &sam_hdr_destroy); + if (!h) + throw std::system_error(std::make_error_code(std::errc(errno)), + "failed to read header: " + filename); + // NOLINTNEXTLINE(cppcoreguidelines-pro-type-union-access) + std::unique_ptr rec(bam_init1(), &bam_destroy1); + + dups_map_t dups; + std::vector buffer; + int r{}; + std::uint64_t n_reads{}; + const auto max_n_reads = n_unique_multiplier * n_unique; + while (n_reads++ < max_n_reads && std::size(dups) < n_unique && + (r = sam_read1(in.get(), h.get(), rec.get())) >= 0) { + const auto l_qseq = rec->core.l_qseq; + const auto seq = bam_get_seq(rec.get()); + if (std::ssize(buffer) < l_qseq) + buffer.resize(l_qseq); + for (auto i = 0; i < l_qseq; ++i) + buffer[i] = seq_nt16_str[bam_seqi(seq, i)]; + if (bam_is_rev(rec.get())) { + std::reverse(std::begin(buffer), std::begin(buffer) + l_qseq); + std::transform(std::cbegin(buffer), std::cbegin(buffer) + l_qseq, + std::begin(buffer), complem); + } + ++dups[falco_word(std::data(buffer), l_qseq)]; + } + if (r < -1) // error + throw std::system_error(std::make_error_code(std::errc(errno)), + "error reading bam record from: " + filename); + return dups; +} + +[[nodiscard]] auto +init_dups_fq(const std::string &filename, const std::uint64_t n_unique) + -> dups_map_t { + static constexpr auto n_unique_multiplier = 10; + const std::unique_ptr in( + bgzf_open(std::data(filename), "r"), &bgzf_close); + if (!in) + throw std::system_error(std::make_error_code(std::errc(errno)), + "failed to open file: " + filename); + dups_map_t dups; + kstring_t s = KS_INITIALIZE; + int r{}; + std::uint64_t n_lines{}; + std::uint64_t n_reads{}; + const auto max_n_reads = n_unique_multiplier * n_unique; + while (n_reads < max_n_reads && std::size(dups) < n_unique && + (r = bgzf_getline(in.get(), '\n', &s)) >= 0) { + if (n_lines % 4 == 1) { + const auto l_qseq = ks_len(&s); + const auto seq = ks_c_str(&s); + ++dups[falco_word(seq, l_qseq)]; + ++n_reads; + } + ++n_lines; + } + if (r < -1) // error + throw std::system_error(std::make_error_code(std::errc(errno)), + "error reading bam record from: " + filename); + ks_free(&s); + return dups; +} + +[[nodiscard]] auto +initialize_original_duplicates(const std::vector &infiles, + const std::vector &infos, + const std::uint32_t n_threads) + -> std::vector { assert(std::size(infiles) == std::size(infos)); const std::uint64_t n_files = std::size(infiles); std::vector dups(n_files); From d798f58b960f7834eb626ddf3831ad73b67b47ca Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:45:49 -0800 Subject: [PATCH 12/22] src/fastq_bgzf_file.hcpp and src/bam_file.hcpp: moving code for initializing original duplication data structures to original_duplicates.cpp --- src/bam_file.cpp | 46 ----------------------------------------- src/bam_file.hpp | 4 ---- src/fastq_bgzf_file.cpp | 32 ---------------------------- src/fastq_bgzf_file.hpp | 4 ---- 4 files changed, 86 deletions(-) diff --git a/src/bam_file.cpp b/src/bam_file.cpp index 90533a8..d160a56 100644 --- a/src/bam_file.cpp +++ b/src/bam_file.cpp @@ -65,52 +65,6 @@ estimate_n_reads_bam(const std::string &filename) return {n_reads_estimate, read_len_estimate, filesize}; } -[[nodiscard]] auto -init_dups(const std::string &filename, const std::uint64_t n_unique) - -> dups_map_t { - static constexpr auto complem = [](const auto x) { - return "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; - }; - static constexpr auto n_unique_multiplier = 10; - const std::unique_ptr in( - hts_open(std::data(filename), "r"), &hts_close); - if (!in) - throw std::system_error(std::make_error_code(std::errc(errno)), - "failed to open file: " + filename); - const std::unique_ptr h( - sam_hdr_read(in.get()), &sam_hdr_destroy); - if (!h) - throw std::system_error(std::make_error_code(std::errc(errno)), - "failed to read header: " + filename); - // NOLINTNEXTLINE(cppcoreguidelines-pro-type-union-access) - std::unique_ptr rec(bam_init1(), &bam_destroy1); - - dups_map_t dups; - std::vector buffer; - int r{}; - std::uint64_t n_reads{}; - const auto max_n_reads = n_unique_multiplier * n_unique; - while (n_reads++ < max_n_reads && std::size(dups) < n_unique && - (r = sam_read1(in.get(), h.get(), rec.get())) >= 0) { - const auto l_qseq = rec->core.l_qseq; - const auto seq = bam_get_seq(rec.get()); - if (std::ssize(buffer) < l_qseq) - buffer.resize(l_qseq); - for (auto i = 0; i < l_qseq; ++i) - buffer[i] = seq_nt16_str[bam_seqi(seq, i)]; - if (bam_is_rev(rec.get())) { - std::reverse(std::begin(buffer), std::begin(buffer) + l_qseq); - std::transform(std::cbegin(buffer), std::cbegin(buffer) + l_qseq, - std::begin(buffer), complem); - } - ++dups[falco_word(std::data(buffer), l_qseq)]; - } - if (r < -1) // error - throw std::system_error(std::make_error_code(std::errc(errno)), - "error reading bam record from: " + filename); - return dups; -} - auto bam_file::load_next(const std::int32_t file_id, task_queue &tq, diff --git a/src/bam_file.hpp b/src/bam_file.hpp index 97a2473..10c5458 100644 --- a/src/bam_file.hpp +++ b/src/bam_file.hpp @@ -129,10 +129,6 @@ class bam_file { estimate_n_reads_bam(const std::string &filename) -> std::tuple; -[[nodiscard]] auto -init_dups(const std::string &filename, const std::uint64_t n_unique) - -> dups_map_t; - inline auto make_tasks(bam_file &reads_file, const std::int64_t n_threads, diff --git a/src/fastq_bgzf_file.cpp b/src/fastq_bgzf_file.cpp index 719e928..0c3605c 100644 --- a/src/fastq_bgzf_file.cpp +++ b/src/fastq_bgzf_file.cpp @@ -55,38 +55,6 @@ estimate_n_reads_fastq_bgzf(const std::string &filename) return {static_cast(n_reads_est), read_len_est, filesize}; } -[[nodiscard]] auto -init_dups_fq(const std::string &filename, const std::uint64_t n_unique) - -> dups_map_t { - static constexpr auto n_unique_multiplier = 10; - const std::unique_ptr in( - bgzf_open(std::data(filename), "r"), &bgzf_close); - if (!in) - throw std::system_error(std::make_error_code(std::errc(errno)), - "failed to open file: " + filename); - dups_map_t dups; - kstring_t s = KS_INITIALIZE; - int r{}; - std::uint64_t n_lines{}; - std::uint64_t n_reads{}; - const auto max_n_reads = n_unique_multiplier * n_unique; - while (n_reads < max_n_reads && std::size(dups) < n_unique && - (r = bgzf_getline(in.get(), '\n', &s)) >= 0) { - if (n_lines % 4 == 1) { - const auto l_qseq = ks_len(&s); - const auto seq = ks_c_str(&s); - ++dups[falco_word(seq, l_qseq)]; - ++n_reads; - } - ++n_lines; - } - if (r < -1) // error - throw std::system_error(std::make_error_code(std::errc(errno)), - "error reading bam record from: " + filename); - ks_free(&s); - return dups; -} - auto fastq_bgzf_file::load_next(const std::int32_t file_id, task_queue &tq, diff --git a/src/fastq_bgzf_file.hpp b/src/fastq_bgzf_file.hpp index 0023291..26a2169 100644 --- a/src/fastq_bgzf_file.hpp +++ b/src/fastq_bgzf_file.hpp @@ -128,10 +128,6 @@ class fastq_bgzf_file { estimate_n_reads_fastq_bgzf(const std::string &filename) -> std::tuple; -[[nodiscard]] auto -init_dups_fq(const std::string &filename, const std::uint64_t n_unique) - -> dups_map_t; - inline auto make_tasks(fastq_bgzf_file &reads_file, const std::int64_t n_threads, From 260869f3f63b2fbd9ca4b87a0a77d51f43f17469 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:47:34 -0800 Subject: [PATCH 13/22] src/original_duplicates.cpp: adding forgotten headers --- src/original_duplicates.cpp | 16 ++++++++++------ 1 file changed, 10 insertions(+), 6 deletions(-) diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index 6fd00e8..7f369a3 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -8,6 +8,10 @@ #include "boost/boost_unordered.hpp" +#include // IWYU pragma: keep +#include +#include + #include #include #include @@ -19,7 +23,7 @@ #include [[nodiscard]] auto -init_dups(const std::string &filename, const std::uint64_t n_unique) +init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { static constexpr auto complem = [](const auto x) { return "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; @@ -65,7 +69,7 @@ init_dups(const std::string &filename, const std::uint64_t n_unique) } [[nodiscard]] auto -init_dups_fq(const std::string &filename, const std::uint64_t n_unique) +init_orig_dups_fq(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { static constexpr auto n_unique_multiplier = 10; const std::unique_ptr in( @@ -123,10 +127,10 @@ initialize_original_duplicates(const std::vector &infiles, } dups[file_id] = falco::is_mapped_reads(infos[file_id].format) - ? init_dups(infiles[file_id], - duplication_results::max_reads_to_hash) - : init_dups_fq(infiles[file_id], - duplication_results::max_reads_to_hash); + ? init_orig_dups_bam(infiles[file_id], + duplication_results::max_reads_to_hash) + : init_orig_dups_fq(infiles[file_id], + duplication_results::max_reads_to_hash); } }); std::ranges::for_each(workers, [](auto &w) { w.join(); }); From bbd4785a550375436d42a55a2149bb814f25e161 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:55:14 -0800 Subject: [PATCH 14/22] src/original_duplicates.cpp: adding to_acgtn in both the init_orig_dups_bam and init_orig_dups_fq to convert all non-ACGT letters to N prior to forming a falco_word when initializing the unique 'reads' for original duplicates analysis --- src/original_duplicates.cpp | 26 ++++++++++++++++++++++---- 1 file changed, 22 insertions(+), 4 deletions(-) diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index 7f369a3..f25d54a 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -25,10 +25,19 @@ [[nodiscard]] auto init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { - static constexpr auto complem = [](const auto x) { - return "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; - }; + // ADS: n_unique_multiplier: contributes to max reads to examine, multiplying + // the n_unique that is the target; impossible to know how many needed to hit + // the target, so try to overshoot. static constexpr auto n_unique_multiplier = 10; + static constexpr auto complem = [](char &x) { + assert(x >= 'A' && x <= 'T'); + x = "TNGNNNCNNNNNNNNNNNNA"[x - 'A']; + }; + static constexpr auto to_acgtn = [](const char x) { + static constexpr char mask = 15; + static constexpr std::span seq_nt16_str_acgtn = "NACNGNNNTNNNNNNN"; + return seq_nt16_str_acgtn[x & mask]; + }; const std::unique_ptr in( hts_open(std::data(filename), "r"), &hts_close); if (!in) @@ -54,7 +63,7 @@ init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) if (std::ssize(buffer) < l_qseq) buffer.resize(l_qseq); for (auto i = 0; i < l_qseq; ++i) - buffer[i] = seq_nt16_str[bam_seqi(seq, i)]; + buffer[i] = to_acgtn(bam_seqi(seq, i)); if (bam_is_rev(rec.get())) { std::reverse(std::begin(buffer), std::begin(buffer) + l_qseq); std::transform(std::cbegin(buffer), std::cbegin(buffer) + l_qseq, @@ -71,7 +80,15 @@ init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) [[nodiscard]] auto init_orig_dups_fq(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { + // ADS: n_unique_multiplier: contributes to max reads to examine, multiplying + // the n_unique that is the target; impossible to know how many needed to hit + // the target, so try to overshoot. static constexpr auto n_unique_multiplier = 10; + static constexpr auto to_acgtn = [](const auto c) { + static constexpr auto mask = 31; + static constexpr std::span to_acgtn_sp = "NANCNNNGNNNNNNNNNNNNTNNNNNNNNNNN"; + return to_acgtn_sp[c & mask]; + }; const std::unique_ptr in( bgzf_open(std::data(filename), "r"), &bgzf_close); if (!in) @@ -87,6 +104,7 @@ init_orig_dups_fq(const std::string &filename, const std::uint64_t n_unique) (r = bgzf_getline(in.get(), '\n', &s)) >= 0) { if (n_lines % 4 == 1) { const auto l_qseq = ks_len(&s); + std::ranges::transform(std::span(s.s, l_qseq), s.s, to_acgtn); const auto seq = ks_c_str(&s); ++dups[falco_word(seq, l_qseq)]; ++n_reads; From 87bfa4cd9a385bfa08d2afd7f39fef719a681f19 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 16:56:38 -0800 Subject: [PATCH 15/22] CMakeLists.txt: original_duplicates object links to HTSLIB so that the htslib headers will be visibile when making original_duplicates.o --- CMakeLists.txt | 2 ++ 1 file changed, 2 insertions(+) diff --git a/CMakeLists.txt b/CMakeLists.txt index b4c0948..c7a7d41 100644 --- a/CMakeLists.txt +++ b/CMakeLists.txt @@ -130,6 +130,8 @@ target_link_libraries(falco_file_format PRIVATE HTSLIB::HTSLIB) target_link_libraries(tile_processor PRIVATE HTSLIB::HTSLIB) target_link_libraries(quality_score PRIVATE HTSLIB::HTSLIB) target_link_libraries(fastq_gz_file PRIVATE HTSLIB::HTSLIB) +target_link_libraries(original_duplicates PRIVATE HTSLIB::HTSLIB) + if(USE_ISAL) target_link_libraries(fastq_gz_file PUBLIC ISAL::ISAL) endif() From d38c6a442ebf6a62c0d09a7d9a1ef7d13291aedb Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 17:00:44 -0800 Subject: [PATCH 16/22] src/original_duplicates.cpp: fix changing transform to for_each_n when complementing the already reversed read sequence for revcomp mappers --- src/original_duplicates.cpp | 3 +-- 1 file changed, 1 insertion(+), 2 deletions(-) diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index f25d54a..66c71da 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -66,8 +66,7 @@ init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) buffer[i] = to_acgtn(bam_seqi(seq, i)); if (bam_is_rev(rec.get())) { std::reverse(std::begin(buffer), std::begin(buffer) + l_qseq); - std::transform(std::cbegin(buffer), std::cbegin(buffer) + l_qseq, - std::begin(buffer), complem); + std::for_each_n(std::begin(buffer), l_qseq, complem); } ++dups[falco_word(std::data(buffer), l_qseq)]; } From 24c9995ed848e1953c5e30e5c49bc487f55d0286 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Sun, 1 Nov 2026 17:43:16 -0800 Subject: [PATCH 17/22] static analysis --- src/bam_file.cpp | 2 -- src/bam_file.hpp | 1 - src/duplication_results.cpp | 3 +++ src/fastq_bgzf_file.cpp | 4 +--- src/fastq_bgzf_file.hpp | 1 - src/original_duplicates.cpp | 8 +++++++- 6 files changed, 11 insertions(+), 8 deletions(-) diff --git a/src/bam_file.cpp b/src/bam_file.cpp index d160a56..b8a28f7 100644 --- a/src/bam_file.cpp +++ b/src/bam_file.cpp @@ -2,9 +2,7 @@ #include "bam_file.hpp" #include "bamrec.hpp" -#include "duplication_results.hpp" #include "falco_utils.hpp" -#include "falco_word.hpp" #include "task_queue.hpp" #include // IWYU pragma: keep diff --git a/src/bam_file.hpp b/src/bam_file.hpp index 10c5458..f781c89 100644 --- a/src/bam_file.hpp +++ b/src/bam_file.hpp @@ -6,7 +6,6 @@ #include "bam_header.hpp" #include "bgzf_block.hpp" #include "bgzf_reader.hpp" -#include "duplication_results.hpp" #include #include diff --git a/src/duplication_results.cpp b/src/duplication_results.cpp index b5a3f0d..ae1f616 100644 --- a/src/duplication_results.cpp +++ b/src/duplication_results.cpp @@ -27,6 +27,7 @@ #include #include #include +#include #include #include #include @@ -238,6 +239,8 @@ duplication_results::get_dups_summary(const std::uint64_t n_reads) const std::vector hist_dedup(max_dup + 1); for (const auto n_copies : std::views::values(dups)) ++hist_dedup[n_copies]; + if (hist_dedup[0] > 0) + throw std::runtime_error("Non-zero count of indexed reads seen 0 times"); for (auto [idx, val] : falco::views::enumerate(hist_dedup)) val = static_cast( get_corrected_count(count_at_limit, n_reads, idx, val)); diff --git a/src/fastq_bgzf_file.cpp b/src/fastq_bgzf_file.cpp index 0c3605c..68536e5 100644 --- a/src/fastq_bgzf_file.cpp +++ b/src/fastq_bgzf_file.cpp @@ -1,15 +1,13 @@ // SPDX-License-Identifier: MIT; Copyright 2026 Andrew D Smith #include "fastq_bgzf_file.hpp" -#include "duplication_results.hpp" #include "falco_utils.hpp" -#include "falco_word.hpp" #include "fqrec.hpp" #include "task_queue.hpp" #include #include -#include +#include // IWYU pragma: keep #include #include diff --git a/src/fastq_bgzf_file.hpp b/src/fastq_bgzf_file.hpp index 26a2169..8656e2d 100644 --- a/src/fastq_bgzf_file.hpp +++ b/src/fastq_bgzf_file.hpp @@ -5,7 +5,6 @@ #include "bgzf_block.hpp" #include "bgzf_reader.hpp" -#include "duplication_results.hpp" #include #include diff --git a/src/original_duplicates.cpp b/src/original_duplicates.cpp index 66c71da..44629d5 100644 --- a/src/original_duplicates.cpp +++ b/src/original_duplicates.cpp @@ -4,24 +4,29 @@ #include "duplication_results.hpp" #include "falco_file_format.hpp" +#include "falco_word.hpp" #include "file_info.hpp" #include "boost/boost_unordered.hpp" #include // IWYU pragma: keep -#include #include #include #include +#include #include +#include #include #include +#include #include +#include #include #include // for std::move #include +namespace { [[nodiscard]] auto init_orig_dups_bam(const std::string &filename, const std::uint64_t n_unique) -> dups_map_t { @@ -116,6 +121,7 @@ init_orig_dups_fq(const std::string &filename, const std::uint64_t n_unique) ks_free(&s); return dups; } +} // namespace [[nodiscard]] auto initialize_original_duplicates(const std::vector &infiles, From 64e610f371a77cf0bf480aad6750c412a5f6c654 Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Mon, 9 Nov 2026 16:05:40 -0800 Subject: [PATCH 18/22] data/test_scripts/iupac_dups.sh: adding tests for dups with iupac symbols ensuring all are treated like N --- data/test_scripts/iupac_dups.sh | 30 ++++++++++++++++++++++++++++++ 1 file changed, 30 insertions(+) create mode 100644 data/test_scripts/iupac_dups.sh diff --git a/data/test_scripts/iupac_dups.sh b/data/test_scripts/iupac_dups.sh new file mode 100644 index 0000000..392fb59 --- /dev/null +++ b/data/test_scripts/iupac_dups.sh @@ -0,0 +1,30 @@ +#!/usr/bin/env bash +# SPDX-License-Identifier: MIT + +# Test that IUPAC symbols are counted as N + +# Need samtools to check that this works for sam/bam formats +if [[ ! -e $(type -P samtools) ]]; then + echo "samtools not found; skipping remaining tests"; + exit 77; +fi + +prog=./falco +infile1=test_data/iupac_dups.fq.gz +infile2=iupac_dups_bam.bam +outdir=iupac_dups_out +if [[ -e "${infile1}" ]]; then + ${prog} --orig-dups -o ${outdir} ${infile1} + samtools view -h -o ${outdir}/${infile2} ${infile1} + ${prog} --orig-dups -o ${outdir} ${outdir}/${infile2} + x=$(md5sum --ignore-missing -c test_data/md5sum.txt | \ + grep "${outdir}" | \ + grep -c "OK$") + if [[ "${x}" != "2" ]]; then + exit 1; + fi + rm -r ${outdir} +else + echo "${infile} not found; skipping remaining tests"; + exit 77; +fi From 6bd1da98eb56eae92f39b8d22fba9e5c6918582b Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Mon, 9 Nov 2026 16:06:18 -0800 Subject: [PATCH 19/22] cmake/tests.cmake: adding tests for IUPAC symbols and duplicates ensuring all non-ACGT are treated like N --- cmake/tests.cmake | 1 + 1 file changed, 1 insertion(+) diff --git a/cmake/tests.cmake b/cmake/tests.cmake index aac54a0..3097ff0 100644 --- a/cmake/tests.cmake +++ b/cmake/tests.cmake @@ -38,3 +38,4 @@ add_test(NAME "FASTQ from stdin" COMMAND bash test_scripts/fastq_stdin.sh) add_test(NAME "SAM from stdin" COMMAND bash test_scripts/sam_stdin.sh) add_test(NAME "Orig GC content" COMMAND bash test_scripts/fastq_orig_gc.sh) add_test(NAME "IUPAC symbols" COMMAND bash test_scripts/fastq_iupac.sh) +add_test(NAME "IUPAC and orig dups" COMMAND bash test_scripts/iupac_dups.sh) From ec7f010915250c8fb78ae5110f476fd7674260eb Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Mon, 9 Nov 2026 16:06:47 -0800 Subject: [PATCH 20/22] data/test_data/md5sum.txt: adding test hashes --- data/test_data/md5sum.txt | 2 ++ 1 file changed, 2 insertions(+) diff --git a/data/test_data/md5sum.txt b/data/test_data/md5sum.txt index 0987ae9..7fcce8d 100644 --- a/data/test_data/md5sum.txt +++ b/data/test_data/md5sum.txt @@ -17,3 +17,5 @@ e786bd4d9e6065904ab7b45021e7190b groups_out/bam_1/fastqc_data.txt bcea1e9f86f0917496b14ae81b6ecbc5 sam_stdin_out/sam_stdin/fastqc_data.txt 088c04f706f340f058b663d6e0209dca fastq_orig_gc_out/fastq_bgzip_1/fastqc_data.txt e71cef09fd4b6f4019f8fdb82ee2eaf3 fastq_iupac_out/fastq_1_iupac/fastqc_data.txt +c827b3a42f08404ac87d3cfb1dc51938 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zM;E-l0Ph9eKJ(2-cIhouvH1T0zK%hn{Gb2;ABzYC000000RIL6LPG)o8vp|U00000 H00000EGlIH literal 0 HcmV?d00001 From 3b02e59f58229bcd92efab9bca7fe0b0920babba Mon Sep 17 00:00:00 2001 From: Andrew D Smith Date: Mon, 9 Nov 2026 16:11:04 -0800 Subject: [PATCH 22/22] data/test_scripts/iupac_dups.sh: fixing typo --- data/test_scripts/iupac_dups.sh | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/data/test_scripts/iupac_dups.sh b/data/test_scripts/iupac_dups.sh index 392fb59..1b34708 100644 --- a/data/test_scripts/iupac_dups.sh +++ b/data/test_scripts/iupac_dups.sh @@ -25,6 +25,6 @@ if [[ -e "${infile1}" ]]; then fi rm -r ${outdir} else - echo "${infile} not found; skipping remaining tests"; + echo "${infile1} not found; skipping remaining tests"; exit 77; fi