fraq: A high-throughput extensible toolkit for processing fastq data ================ `fraq` is a high-throughput extensible toolkit for processing fastq data. The goal of this package is to empower users to quickly build out programmatic ‘kernels’ to define any FASTQ processing task they may need. `fraq` then takes those kernels and handles I/O, compression and multithreading. It builds on Intel TBB’s flow graph to orchestrate concurrency and data processing; throughput can be as fast as compression and disk speed allow. The package ships with a suite of predefined ‘kernels’ for common FASTQ tasks, detailed in this vignette. ## Why use fraq? - **Concurrent I/O and data processing.** As fast as compression and disk speed allow - **Extensibility.** You can drop in your own kernels for almost any fastq processing task (see `Extension system` sections below) - **Support for gzip, zstd and pipes.** See `Supported formats` section ## Quick start **Install** ``` r if (!requireNamespace("BiocManager", quietly = TRUE)) { install.packages("BiocManager") } BiocManager::install("fraq") ``` ## Pre-built functions fraq ships with a collection of ready-to-go kernels that cover common preprocessing steps: - `fraq_downsample()` - deterministically retain a target fraction of reads (paired-end aware) - `fraq_convert()` - re-encode reads between any supported formats - `fraq_concat()` - glue multiple inputs together - `fraq_chunk()` - split streams into chunk-numbered files with a suffix-driven format - `fraq_slice()` - keep the first `limit` reads or select specific read indices - `fraq_count_barcodes()` - tally barcode hits with optional reverse-complement handling - `fraq_demux()` - shard reads into files derived from a `{barcode}` placeholder - `fraq_quality_filter()` - drop read groups that fail read quality thresholds - `fraq_merge_pairs()` - overlap paired reads to create consensus single-end reads while recording merge stats - `fraq_trim_adapters()` - trim adapters from the first mate (and optionally drop untrimmed records) - `fraq_summary()` - compute per-base stats, histograms, and insert-size estimates ## Walkthroughs with synthetic data For illustration purposes, we create a small synthetic dataset. ``` r set.seed(314156) example_dir <- file.path(tempdir(), "fraq_function_examples") dir.create(example_dir, showWarnings = FALSE, recursive = TRUE) R1 <- file.path(example_dir, "example_R1.fastq") R2 <- file.path(example_dir, "example_R2.fastq") generate_random_fastq(c(R1, R2), n_reads = 2000, read_length = 150) example_reads <- c(R1, R2) ``` ### Summarization `fraq_summary()` rolls up QC tables for the R1/R2 fastq pairs. ``` r library(fraq) # summarize quality metrics qc <- fraq_summary(c(R1, R2)) ``` <figure> <img src="vignettes/plots/fig-qc-quicklook-1.png" alt="Quick QC overview from fraq_summary()." /> <figcaption aria-hidden="true">Quick QC overview from <code>fraq_summary()</code>.</figcaption> </figure> ### Filtering Various filtering operations are illustrated below. Here we downsample to reduce dataset size and then discard mates whose mean PHRED drops below 22 or accumulate more than 6 bases with PHRED \< 18 (roughly 10% of reads will be filtered). ``` r filter_dir <- file.path(example_dir, "filtering") dir.create(filter_dir, showWarnings = FALSE) downsampled_reads <- file.path( filter_dir, c("example_R1_ds.fastq.gz", "example_R2_ds.fastq.gz") ) fraq_downsample(example_reads, downsampled_reads, amount = 0.50, nthreads = 2L) quality_reads <- file.path( filter_dir, c("example_R1_q20.fastq.gz", "example_R2_q20.fastq.gz") ) fraq_quality_filter( input = downsampled_reads, output = quality_reads, min_mean_quality = 22, max_low_q_bases = 6L, low_q_threshold = 18L, nthreads = 2L ) filtered_stats <- fraq_summary(quality_reads, nthreads = 2L)$basic_stats_R1 filtered_stats ``` ## total_sequences total_bases seq_len_min seq_len_mean seq_len_max gc_percent ## 1 970 145500 150 150 150 50.00619 ### Splitting Splitting workflows either direct reads into barcode-specific files, chunk long runs into bite-sized batches, or simply inspect barcode usage. ``` r split_dir <- file.path(example_dir, "splitting") if (dir.exists(split_dir)) unlink(split_dir, recursive = TRUE) dir.create(split_dir, showWarnings = FALSE) barcode_set <- c("ACGTAA", "TTGGCC") demux_patterns <- file.path( split_dir, c("R1_{barcode}.fastq.gz", "R2_{barcode}.fastq.gz") ) ``` **Barcode-guided demultiplexing** Demux looks for barcode/adapter/primer sequence at the start of the first given fastq file. ``` r fraq_demux( input = example_reads, output_format = demux_patterns, barcodes = barcode_set, max_distance = 1L, nthreads = 2L ) basename(sort( list.files(split_dir, pattern = "R1_.*\\.fastq.gz$", full.names = TRUE) )) ``` ## [1] "R1_ACGTAA.fastq.gz" "R1_NO_MATCH.fastq.gz" "R1_TTGGCC.fastq.gz" **Fixed-size chunking** `fraq_chunk` splits reads into fixed-size batches with incremental file names. ``` r chunk_prefix <- file.path(split_dir, "chunk_demo") fraq_chunk( input = example_reads[1], output_prefix = chunk_prefix, output_suffix = "gz", chunk_size = 200, nthreads = 2L ) basename(sort( list.files( split_dir, pattern = "chunk_demo_chunk.+\\.fastq.gz$", full.names = TRUE ) )) ``` ## [1] "chunk_demo_chunk0.fastq.gz" "chunk_demo_chunk1.fastq.gz" ## [3] "chunk_demo_chunk2.fastq.gz" "chunk_demo_chunk3.fastq.gz" ## [5] "chunk_demo_chunk4.fastq.gz" "chunk_demo_chunk5.fastq.gz" ## [7] "chunk_demo_chunk6.fastq.gz" "chunk_demo_chunk7.fastq.gz" ## [9] "chunk_demo_chunk8.fastq.gz" "chunk_demo_chunk9.fastq.gz" **Barcode counting** Barcode counting looks for sequence substrings anywhere in the fastq reads and outputs a data frame of counts. ``` r fraq_count_barcodes( input = example_reads, barcodes = barcode_set, max_distance = 0L, allow_revcomp = FALSE, nthreads = 2L ) ``` ## barcode count ## 1 NO_MATCH 1734 ## 2 TTGGCC 132 ## 3 ACGTAA 126 ## 4 MULTI_MATCH 8 ### Modification Format conversion, adapter trimming, consensus merging, etc. ``` r mod_dir <- file.path(example_dir, "modification") if (dir.exists(mod_dir)) unlink(mod_dir, recursive = TRUE) dir.create(mod_dir, showWarnings = FALSE) ``` **Convert between formats** Re-wrap the paired-end files in Zstandard-compressed FASTQ. ``` r converted_fastq <- file.path( mod_dir, c("example_R1.fastq.zst", "example_R2.fastq.zst") ) fraq_convert(example_reads, converted_fastq, nthreads = 2L) ``` **Concatenate files** Combine the converted shards into a single gzip-compressed stream. ``` r concatenated <- file.path(mod_dir, "example_all.fastq.gz") fraq_concat(converted_fastq, concatenated, nthreads = 2L) ``` **Merge overlapping pairs** Generate consensus single-end reads while keeping optional unmerged outputs. ``` r merged_reads <- file.path(mod_dir, "example_merged.fastq") unmerged_reads <- file.path( mod_dir, c("example_unmerged_R1.fastq", "example_unmerged_R2.fastq") ) fraq_merge_pairs( input = example_reads, output_merged = merged_reads, output_unmerged = unmerged_reads, min_overlap = 20L, max_mismatch_rate = 0.05, nthreads = 2L ) ``` ## $merged_reads ## [1] 648 ## ## $unmerged_reads ## [1] 1352 ## ## $mean_insert_size ## [1] 267.7454 ## ## $sd_insert_size ## [1] 10.25771 ## ## $mean_overlap ## [1] 32.25463 ## ## $mean_mismatch_rate ## [1] 0 **Trim adapters** Remove adapter prefixes from R1 (and optionally drop untrimmed reads). ``` r trimmed_reads <- file.path( mod_dir, c("example_trimmed_R1.fastq", "example_trimmed_R2.fastq") ) fraq_trim_adapters( input = example_reads, output = trimmed_reads, adapters = "ACTAC", max_distance = 1L, filter_untrimmed = FALSE, nthreads = 2L ) ``` ## adapter count ## 1 NO_ADAPTER 1973 ## 2 ACTAC 27 ## Supported formats fraq chooses formats from file names, so the extension you supply controls how data is decoded and encoded. - `.fastq`, `.fq` - Plain FASTQ (read/write). Fastest path but no compression. - `.fastq.gz` - Gzip-compressed FASTQ (read/write). Uses zlib; tune via `fraq_options("gzip_compress_level")`. - `.fastq.zst` - Zstd-compressed FASTQ (read/write). Uses bundled zstd; tune via `fraq_options("zstd_compress_level")`. - `.fraq` - Chunked binary container (read/write). Designed for multithreaded IO and compression; see [FRAQ file format](#fraq-file-format) for layout details. FRAQ is a binary, block-compressed format layered on top of bundled zstd so it is both more storage-efficient and faster to stream than textual FASTQ. - `.mem` - In-memory `.fraq` (read/write). Lifetime is limited to the current R session. - `.fifo` - POSIX named pipes on Linux/macOS (read/write). Useful for streaming data between CLI programs. ## Extension system: flow graph overview The `fraq_run()` pipeline wires a TBB flow graph so that IO and data flow happen concurrently with data processing. A high-level view looks like this:  Each block of fastq is read from `Primary Reader` (for the first mate R1) and `Secondary Readers` (for R2 or additional fastq files). The key node in this graph is the `Process Kernel`. It takes fastq records (i.e. R1 and R2 reads), processes or alters them, decides whether to keep them or not (filtering) and outputs any number of fastq records (demux and splitting). This simple pattern naturally supports lots of different fastq processing operations and can be customized. ## Extension system: writing a custom kernel with Rcpp If the prebuilt kernels are insufficient, you can write your own via an Rcpp script. You supply a lambda to `fraq::run`, and the runtime handles all batching, IO, and parallelism. More information can be found in `?fraq_rcpp_template`. Below is an example that keeps only reads whose GC fraction falls in a window (default 35-65%). ``` cpp // [[Rcpp::depends(fraq)]] #include <Rcpp.h> #include <fraq.h> double calc_gc_content(const std::string &s) { double gc = 0.0; for(char c : s) { if(c == 'G' || c == 'C') gc += 1.0 ; } return gc / (double) s.size(); } // [[Rcpp::export(rng=false)]] void fraq_gc_filter(std::vector<std::string> input, std::vector<std::string> output, double gc_min = 0.35, double gc_max = 0.65) { auto gc_filter_kernel = [&](fraq::input_t reads, size_t read_index) -> fraq::output_t { for(auto & read : reads) { double gc = calc_gc_content(read.seq); if(gc < gc_min || gc > gc_max) return {}; } return fraq::zip(output, std::move(reads)); }; fraq::FraqRunConfig cfg; cfg.zstd_compress_level = 5; int nthreads = 4; fraq::run(input, gc_filter_kernel, nthreads, cfg); } ``` All `fraq` classes are transparent structures with no private members, built on standard library types. - A `Read` is a struct with three strings: `name`, `seq`, and `qual` containing read info for a single mate - `input_t` is a vector of `Read`’s representing a single fastq record (i.e. R1 and R2 reads) - `output_t` is a `std::vector` of output paths / Reads Compile via `Rcpp::sourceCpp()` then in R you can call your custom kernel as a normal function. The extensions on the output paths decide output format automatically. ``` r input <- c("sample_R1.fastq.gz", "sample_R2.fastq.gz") output <- c("filtered_R1.fastq.zst", "filtered_R2.fastq.zst") fraq_gc_filter(input, output, gc_min = 0.30, gc_max = 0.70) ``` ### Some important tips when building custom kernels - You are still responsible for writing safe C++ - Avoid interacting with the R API or relying on `Rcpp` classes; if your kernel interacts with R, you **must** use `nthreads = 1` ### Streaming with named pipes You can use named pipes (Linux/Mac only - Windows is not supported) to stream input and output directly into other command line programs. Below is an example using `fraq_downsample` on input fastqs (random fastqs in this example) and streaming the output directly to `bwa-mem2`. **downsample_fifo.R** ``` r library(fraq) generate_random_fastq("R1.fastq") generate_random_fastq("R2.fastq") fraq_downsample(input=c("R1.fastq", "R2.fastq"), output=c("ds_R1.fastq.fifo", "ds_R2.fastq.fifo"), amount = 0.25, nthreads = 5L) ``` In bash (Linux/macOS), create the named pipes first before any operations: ``` bash HG38_REF=/path/to/hg38.fa.gz mkfifo ds_R1.fastq.fifo ds_R2.fastq.fifo Rscript downsample_fifo.R & bwa-mem2 mem -t 8 $HG38_REF ds_R1.fastq.fifo ds_R2.fastq.fifo > output.sam ``` > Windows users should stick with regular files or platform-specific > piping; `.fifo` paths are not available there. ## Tuning and threading Global knobs live behind `fraq_options()`: ``` r fraq_options("blocksize") # current block size (default: 65535 reads) ``` ## [1] 65535 ``` r fraq_options("blocksize", 16384L) # shrink batches when running small tests ``` ## [1] 65535 ``` r fraq_options("zstd_compress_level", 6L) ``` ## [1] 3 ``` r fraq_options("gzip_compress_level", 4L) ``` ## [1] 6 Each kernel also accepts `nthreads`. Internally, fraq caps the TBB scheduler to the requested parallelism. ## FRAQ file format The `.fraq` container stores FASTQ reads in independent blocks so that IO and block-level compression can run concurrently. Each block holds up to 65,535 reads (`fraq_options("blocksize")`) and stores block-level info such as zstd compression, name-prefix factoring, and optional nucleotide bit-packing. FRAQ takes inspiration from the Nucleotide Archival Format (NAF) by concatenating nucleotide and quality payloads before compressing them with zstd (improving compression efficiency), following the approach described by Kryukov et al. (2019). FRAQ differs by block compressing the stream, which enables multithreaded compression, streaming and tailoring the layout specifically to the FASTQ format instead of being more general. **Specification** - **File header (16 bytes).** Bytes 1-4 are the ASCII magic `FRAQ`. Bytes 5-14 are reserved (currently zero). Byte 15 records the writer’s endianness (mixed endianness is rejected in v1), and byte 16 is the format version (currently `0x01`). - **Block header.** Every block begins with a 32-bit metadata word that packs 11 two-bit codes plus a single bit for `use_bit_pack`. The codes describe the byte-width (1/2/4/8) used for each scalar that follows so the block stays compact regardless of read count or payload size. - **Scalar section.** After the metadata word come nine unsigned integers whose widths are dictated by the codes: `num_reads`, `uncompressed_names_size`, `uncompressed_seqs_size`, `name_prefix_size`, and the byte sizes of the five compressed buffers (`compressed_name_lengths`, `compressed_names`, `compressed_seq_lengths`, `compressed_seqs`, `compressed_quals`). - **Payload order.** The payload immediately follows the scalars: 1. `name_prefix` - a raw string that all reads share (for example the sample identifier preceding `/1` or `/2`). 2. `compressed_name_lengths` - zstd-compressed array of per-read tail lengths. 3. `compressed_names` - concatenated name tails compressed with zstd. 4. `compressed_seq_lengths` - zstd-compressed per-read sequence lengths. 5. `compressed_seqs` - either raw bases or 4-bit packed codes (A/C/G/T/R/Y/S/W/K/M/B/D/H/V/N/U) compressed with zstd. 6. `compressed_quals` - zstd-compressed ASCII Phred strings; qualities are never bit-packed. - **Decoding.** Readers expand the zstd streams, rebuild each read by combining `name_prefix` plus the stored tail, and emit the number of sequences indicated by `num_reads`. *Reference:* Kryukov, Kirill, et al. “Nucleotide Archival Format (NAF) enables efficient lossless reference-free compression of DNA sequences.” *Bioinformatics* 35.19 (2019): 3826-3828.