> This page is for version v4.8.0 (default). > For other versions, use one of these documentation indexes: > - v4.8.0 (default): https://docs.nvidia.com/clara/parabricks/v4.8.0/llms.txt > - v4.7.1: https://docs.nvidia.com/clara/parabricks/v4.7.1/llms.txt > For clean Markdown of any page, append .md to the page URL. > For a complete documentation index, see https://docs.nvidia.com/clara/parabricks/llms.txt. > For AI client integration (Claude Code, Cursor, etc.), connect to the MCP server at https://docs.nvidia.com/clara/parabricks/_mcp/server. # fq2bam_meth Generate BAM/CRAM output given one or more pairs of FASTQ files from bisulfite sequencing (BS-Seq). Can also optionally generate a BQSR report. Refer to the [fq2bam_meth Reference](#options) section for a detailed listing of all available options. ## What is fq2bam_meth? The tool fq2bam_meth is a fast, accurate algorithm for mapping methylated DNA sequence reads to a reference genome, performing local alignment, and producing alignment for different parts of the query sequence. It implements the baseline tool bwa-meth [^bwameth-repo] [^bwameth-manuscript] in a performant method using [fq2bam (BWA-MEM + GATK)](/clara/parabricks/v4.8.0/tool-reference/tools/fq2bam) as a backend for processing on GPU. ## Why fq2bam_meth? fq2bam_meth is the Parabricks wrapper for bwa-meth, which will sort the output and can mark duplicates and recalibrate base quality scores in line with GATK best practices. The Parabricks `fq2bam_meth` tool is capable of handling longer reads and is less sensitive to errors than other alignment algorithms. We enable fast and accurate whole-genome bisulfite sequencing (WGBS) to detect DNA-methylation at the single base pair level [^bwameth-doi]. Some of the advantages of using fq2bam_meth over similar tools include: 1. It is faster than many other BS-Seq alignment algorithms, making it the ideal choice for high-throughput analysis. 2. It maintains compatibility with existing CPU-based tools. ## How Should I Use fq2bam_meth? fq2bam_meth uses an accelerated version of BWA-MEM to generate BAM/CRAM output given one or more pairs of FASTQ files from BS-Seq. The user can turn-off marking of duplicates by adding the `--no-markdups` option. The BQSR step is only performed if the `--knownSites input` and `--out-recal-file output` options are provided; doing so will also generate a BQSR report. *Prior* to running alignment, the reference genome must be converted using baseline bwa-meth. The bwa-meth indexing step produces a reference `fasta` file with a name formatted as `fasta.bwameth.c2t`. The indexing preparation step requires running `bwameth.py index $REF.fasta`. Baseline [bwa-meth](https://github.com/brentp/bwa-meth/) requires baseline [BWA-MEM](https://github.com/lh3/bwa) to be in the user's path for indexing functionality. Note that indexing is a time-consuming prerequisite that should only need to be completed once per reference genome. The `bwameth.py` script can be found [here](https://github.com/brentp/bwa-meth/blob/v0.2.7/bwameth.py). ![](/clara/parabricks/_fern-files/_img/8a5cd0cb164cf1c6796a6a90d6f5b147e52a9c6641ecee0f3b1db74e1bd5656e.webp) ## Quick Start ```sh # This command assumes all the inputs are in the current working directory and all the outputs go to the same place. docker run --rm --gpus all --volume $(pwd):/workdir --volume $(pwd):/outputdir \ --workdir /workdir \ nvcr.io/nvidia/clara/clara-parabricks:4.8.0-1 \ pbrun fq2bam_meth \ --ref /workdir/${REFERENCE_FILE} \ --in-fq /workdir/${INPUT_FASTQ_1} /workdir/${INPUT_FASTQ_2} \ --knownSites /workdir/${KNOWN_SITES_FILE} \ --out-bam /outputdir/${OUTPUT_BAM} \ --out-recal-file /outputdir/${OUTPUT_RECAL_FILE} ``` ## Useful Options for Performance Suggested parameters to control host memory use: * The parameter `--bwa-normalized-queue-capacity` controls the amount of batches that will be in memory to be processed. By default, the value normalized to the number of GPUs in the run is 10, corresponding to ` * ` for the total queue size. Multiple queues are used. Lowering this value would be a good strategy to reduce host memory use. However, a value that is too low may hamper performance. By default, Parabricks automatically configures GPU streams and CPU threading. The option `--bwa-nstreams auto` selects the number of streams based on total GPU memory. This heuristic is tuned for a human reference genome; if a run exceeds GPU memory, users should lower `--bwa-nstreams` manually or use `--low-memory`. The option `--bwa-primary-cpus auto` selects 16 primary CPU threads per GPU, while `--bwa-cpu-thread-pool` defaults to 1 thread per primary CPU thread. These defaults are optimized for end-to-end runtime. Users may experiment further with the `--bwa-nstreams`, `--bwa-primary-cpus`, and `--bwa-cpu-thread-pool` parameters to potentially achieve better performance given a particular system configuration. For advanced performance tuning, the options `--bwa-primary-cpus` and `--bwa-cpu-thread-pool` provide fine-grained control of CPU threading. A manually set `--bwa-primary-cpus` value is the total number of primary CPU threads for the whole run, not a per-GPU value; only `auto` scales with the number of GPUs. Each primary CPU thread drives `P` CPU thread pool threads as specified with the option `--bwa-cpu-thread-pool`. The total number of CPU threads processing the CPU stages of alignment is the product of the `--bwa-primary-cpus` and `--bwa-cpu-thread-pool` parameters. This allows the user to control the ratio of "primary" CPU threads, which act independently, to thread pool threads, which act in unison. Depending on the server configuration, input data, and available GPUs, performance may improve by adjusting the balance between primary CPU threads and thread pool threads. Changing the number of primary CPU threads may increase the CPU resources required. * The parameter `--gpuwrite` uses the GPU to compress the final BAM or CRAM file for improved performance during the final stage. * `--gpuwrite-deflate-algo` can be used to control the compression ratio. See below for more details. * The parameter `--gpusort` uses the GPU to sort the BAM or CRAM file. * The option `--cigar-on-gpu` offloads CIGAR generation from the CPU (default) to the GPU. Consider enabling it on CPU-constrained systems, or if benchmarking with and without the option indicates the CPU is a bottleneck; it may improve overall runtime. * The option `--max-read-length-on-gpu` can be set to 1024 bp to process longer reads on GPU. Keep the default value of 512 bp unless the input has many reads longer than 512 bp. Batches with reads longer than the selected value continue to use CPU recovery. * On multi-GPU systems, the option `--run-partition` enables partition mode, which forks one worker process per partition to more efficiently split work across GPUs. We recommend trying this on systems with 4 or more GPUs. The `--bwa-gpu-num-per-partition` option controls how many GPUs each partition drives (default: 2). ## Compatible CPU-based bwa-meth, GATK4 Commands The commands below are the bwa-meth-0.2.7, bwa-0.7.17, and GATK4 counterpart of the Parabricks command above. The output from these commands will be identical to the output from the above command. Refer to the [Output Comparison](/clara/parabricks/v4.8.0/about-parabricks/comparison-with-baseline-tools) page for comparing the results. > **Note** > > Set `--bwa-options="-K 10000000"` in fq2bam_meth and `-K 10000000` in baseline to produce compatible pair-ended results. > **Note** > > fq2bam_meth will not strip `_R1` and `_R2` from read names during preprocessing like baseline bwa-meth. ```sh # Run bwa-meth and pipe the output to create a sorted BAM. $ python bwa-meth.py \ --read-group '@RG\tID:sample_rg1\tLB:lib1\tPL:bar\tSM:sample\tPU:sample_rg1' \ --reference /${REFERENCE_FILE} /${INPUT_FASTQ_1} /${INPUT_FASTQ_2} \ -t 32 -K 10000000 | \ gatk SortSam \ --java-options -Xmx30g \ --MAX_RECORDS_IN_RAM 5000000 \ -I /dev/stdin \ -O cpu.bam \ --SORT_ORDER coordinate # Mark duplicates. $ gatk MarkDuplicates \ --java-options -Xmx30g \ -I cpu.bam \ -O mark_dups_cpu.bam \ -M metrics.txt # Generate a BQSR report. $ gatk BaseRecalibrator \ --java-options -Xmx30g \ --input mark_dups_cpu.bam \ --output /${OUTPUT_RECAL_FILE} \ --known-sites /${KNOWN_SITES_FILE} \ --reference /${REFERENCE_FILE} ``` ## Source of Mismatches While Parabricks `fq2bam_meth` does not lose any accuracy in functionality when compared with BWA-mem and GATK there are several sources that can result in differences in output files. * **BWA-mem** `-K` **argument** In pair-ended mode, the chunk size specified by `-K` can cause small mismatches in the output BAM file. To get rid of the mismatches here, make sure to pass the same number to both baseline BWA-mem and Parabricks `fq2bam_meth`, e.g. `-K 10000000`. * `PA` **aux tag** * Parabricks `fq2bam_meth` puts the `PA` tag last while BWA-mem puts it first. * BWA-mem rounds `PA` tag to 3 digits while Parabricks `fq2bam_meth` does not. The aux tag can be filtered by running `samtools view -x ` * `MC` **(mate-CIGAR) aux tag** * As `fq2bam_meth` is compatible with BWA-MEM 0.7.17, paired-end records now receive an `MC` mate-CIGAR tag. To recapitulate the behavior of BWA-MEM 0.7.15 and earlier by omitting the mate-CIGAR tag, use `--no-mate-cigar`. * **Unmapped reads** * Parabricks `fq2bam_meth` sorts unmapped reads slightly differently than baseline GATK SortSam. Unmapped reads can be filtered with `samtools` by doing `samtools view -F 4`. ## Options | Type | Name | Required? | Description | | ----------- | --------------------------------------------------------------------- | --------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | I/O | `--ref REF` | Yes | Path to the reference file. We will automatically look for \.bwameth.c2t. Converted fasta reference must exist from prior conversion with baseline bwa-meth. | | I/O | `--in-fq [IN_FQ ...]` | No | Path to the pair-ended FASTQ files followed by optional read groups with quotes (Example: "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:foo"). The files must be in fastq or fastq.gz format. All sets of inputs should have a read group; otherwise, none should have a read group, and it will be automatically added by the pipeline. This option can be repeated multiple times. Example 1: --in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz --in-fq sampleX_2_1.fastq.gz sampleX_2_2.fastq.gz. Example 2: --in-fq sampleX_1_1.fastq.gz sampleX_1_2.fastq.gz "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:unit1" --in-fq sampleX_2_1.fastq.gz sampleX_2_2.fastq.gz "@RG\tID:foo2\tLB:lib1\tPL:bar\tSM:sample\tPU:unit2". For the same sample, Read Groups should have the same sample name (SM) and a different ID and PU. | | I/O | `--in-se-fq [IN_SE_FQ ...]` | No | Path to the single-ended FASTQ file followed by optional read group with quotes (Example: "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:foo"). The file must be in fastq or fastq.gz format. Either all sets of inputs have a read group, or none should have one, and it will be automatically added by the pipeline. This option can be repeated multiple times. Example 1: --in-se-fq sampleX_1.fastq.gz --in-se-fq sampleX_2.fastq.gz . Example 2: --in-se-fq sampleX_1.fastq.gz "@RG\tID:foo\tLB:lib1\tPL:bar\tSM:sample\tPU:unit1" --in-se-fq sampleX_2.fastq.gz "@RG\tID:foo2\tLB:lib1\tPL:bar\tSM:sample\tPU:unit2" . For the same sample, Read Groups should have the same sample name (SM) and a different ID and PU. | | I/O | `--in-fq-list IN_FQ_LIST` | No | Path to a file that contains the locations of pair-ended FASTQ files. Each line must contain the location of two FASTQ files followed by a read group, each separated by a space. Each set of files (and associated read group) must be on a separate line. Files must be in fastq/fastq.gz format. Line syntax: \ \ \. | | I/O | `--in-se-fq-list IN_SE_FQ_LIST` | No | Path to a file that contains the locations of single-ended FASTQ files. Each line must contain the location of the FASTQ files followed by a read group, each separated by a space. Each file (and associated read group) must be on a separate line. Files must be in fastq/fastq.gz format. Line syntax: \ \. | | I/O | `--knownSites KNOWNSITES` | No | Path to a database of known polymorphic sites used to exclude regions around known polymorphisms from analysis. The file can be in vcf or vcf.gz format. This option can be used multiple times. | | I/O | `--interval-file INTERVAL_FILE` | No | Path to an interval file in one of these formats: Picard-style (.interval_list or .picard), GATK-style (.list or .intervals), or BED file (.bed). This option can be used multiple times. | | I/O | `--out-recal-file OUT_RECAL_FILE` | No | Path of a report file after Base Quality Score Recalibration. | | I/O | `--out-bam OUT_BAM` | Yes | Path of a BAM/CRAM file. | | I/O | `--out-duplicate-metrics OUT_DUPLICATE_METRICS` | No | Path of duplicate metrics file after marking duplicates. | | I/O | `--out-qc-metrics-dir OUT_QC_METRICS_DIR` | No | Path of the directory where QC metrics will be generated. | | Tool | `--max-read-length MAX_READ_LENGTH` | No | Maximum read length/size (i.e., sequence length) used for bwa and filtering FASTQ input. (default: 480) | | Tool | `--min-read-length MIN_READ_LENGTH` | No | Minimum read length/size (i.e., sequence length) used for bwa and filtering FASTQ input. (default: 1) | | Tool | `--max-read-length-on-gpu MAX_READ_LENGTH_ON_GPU` | No | Maximum read length to process on GPU. Reads longer than this value, up to --max-read-length, will use the CPU fallback. Keep the default value (512 base pairs) unless the input has many reads longer than 512 base pairs; to process longer reads at all, also raise --max-read-length. Valid values are 512 and 1024 base pairs only. | | Tool | `-L INTERVAL, --interval INTERVAL` | No | Interval within which to call bqsr from the input reads. All intervals will have a padding of 100 to get read records, and overlapping intervals will be combined. Interval files should be passed using the --interval-file option. This option can be used multiple times (e.g. "-L chr1 -L chr2:10000 -L chr3:20000+ -L chr4:10000-20000"). | | Tool | `--bwa-options BWA_OPTIONS` | No | Pass supported bwa mem options as one string. The current original bwa mem supported options are: -M, -Y, -C, -T, -B, -U, -L, -I, -S, -P, -5, -q, and -K (e.g. --bwa-options="-M -Y"). | | Tool | `--no-warnings` | No | Suppress warning messages about system thread and memory usage. | | Tool | `--filter-flag FILTER_FLAG` | No | Don't generate SAM entries in the output if the entry's flag's meet this criteria. Criteria: (flag & filter != 0). (default: 0) | | Tool | `--skip-multiple-hits` | No | Filter SAM entries whose length of SA is not 0. | | Tool | `--align-only` | No | Generate output BAM after bwa-mem. The output will not be co-ordinate sorted or duplicates will not be marked. | | Tool | `--no-markdups` | No | Do not perform the Mark Duplicates step. Return BAM after sorting. | | Tool | `--markdups-single-ended-start-end` | No | Mark duplicate on single-ended reads by 5' and 3' end. | | Tool | `--ignore-rg-markdups-single-ended` | No | Ignore read group info in marking duplicates on single-ended reads. This option must be used with `--markdups-single-ended-start-end`. | | Tool | `--fix-mate` | No | Add mate cigar (MC) and mate quality (MQ) tags to the output file. | | Tool | `--no-mate-cigar` | No | Do not add mate CIGAR (MC) tags to the output. Use this option to reproduce the behavior of BWA-MEM versions before 0.7.16, which did not emit MC tags. Cannot be used with `--fix-mate`. | | Tool | `--markdups-assume-sortorder-queryname` | No | Assume the reads are sorted by queryname for marking duplicates. This will mark secondary, supplementary, and unmapped reads as duplicates as well. This flag will not impact variant calling while increasing processing times. | | Tool | `--markdups-picard-version-2182` | No | Assume marking duplicates to be similar to Picard version 2.18.2. | | Tool | `--monitor-usage` | No | Monitor approximate CPU utilization and host memory usage during execution. | | Tool | `--optical-duplicate-pixel-distance OPTICAL_DUPLICATE_PIXEL_DISTANCE` | No | The maximum offset between two duplicate clusters in order to consider them optical duplicates. Ignored if --out-duplicate-metrics is not passed. | | Tool | `--standalone-bqsr` | No | Run standalone BQSR. | | Tool | `--read-group-sm READ_GROUP_SM` | No | SM tag for read groups in this run. | | Tool | `--read-group-lb READ_GROUP_LB` | No | LB tag for read groups in this run. | | Tool | `--read-group-pl READ_GROUP_PL` | No | PL tag for read groups in this run. | | Tool | `--read-group-id-prefix READ_GROUP_ID_PREFIX` | No | Prefix for the ID and PU tags for read groups in this run. This prefix will be used for all pairs of FASTQ files in this run. The ID and PU tags will consist of this prefix and an identifier, that will be unique for a pair of FASTQ files. | | Tool | `-ip INTERVAL_PADDING, --interval-padding INTERVAL_PADDING` | No | Amount of padding (in base pairs) to add to each interval you are including. | | Tool | `--set-as-failed SET_AS_FAILED` | No | Flag alignments to strand 'f' or 'r' as failing quality-control (QC) with the failed QC flag 0x200. BS-Seq libraries are often to a single strand; other strands can be flagged as QC failures. Note: f == OT, r == OB. Valid options are 'f' or 'r'. | | Tool | `--do-not-penalize-chimeras` | No | Turn off the default heuristic which marks alignments as failing QC if the longest match is less than 44% of the original sequence length. Alignments which fail this heuristic are also un-paired. | | Performance | `--bwa-nstreams BWA_NSTREAMS` | No | Number of streams per GPU to use; note: more streams increases device memory usage. Default is auto which will try to use an optimal amount of device memory. (default: auto) | | Performance | `--bwa-cpu-thread-pool BWA_CPU_THREAD_POOL` | No | Number of threads to devote to CPU thread pool *per primary CPU thread*. (default: 1) | | Performance | `--num-cpu-threads-per-stage NUM_CPU_THREADS_PER_STAGE` | No | (Same as above) Number of threads to devote to CPU thread pool *per primary CPU thread*. | | Performance | `--bwa-normalized-queue-capacity BWA_NORMALIZED_QUEUE_CAPACITY` | No | Normalized capacity for alignment work queues, use a lower value if CPU memory is low; final value will be \ \* \. (default: 10) | | Performance | `--bwa-primary-cpus BWA_PRIMARY_CPUS` | No | Total number of primary CPU threads for the run, each driving its own thread pool. Threads are not tied to GPUs. With --run-partition, this total is divided evenly across partitions and must be divisible by the partition count. When set to 'auto', the number of GPUs is used as a heuristic to determine the primary-cpu thread count (16 per GPU) (default: auto) | | Performance | `--cigar-on-gpu` | No | Run CIGAR generation on GPU. Helpful in CPU bound conditions. (default is on CPU). | | Performance | `--run-partition` | No | Divide --num-gpus into partitions and run multiple child processes in parallel, each pinned to its share of GPUs via CUDA_VISIBLE_DEVICES. The number of partitions is --num-gpus / --bwa-gpu-num-per-partition. | | Performance | `--bwa-gpu-num-per-partition BWA_GPU_NUM_PER_PARTITION` | No | Number of GPUs each partition (worker process) drives when --run-partition is set. Default 2. Must divide --num-gpus evenly. | | Performance | `--gpuwrite` | No | Use one GPU to accelerate writing final BAM/CRAM. | | Performance | `--gpuwrite-deflate-algo GPUWRITE_DEFLATE_ALGO` | No | Choose the nvCOMP DEFLATE algorithm to use with --gpuwrite. Note these options do not correspond to CPU DEFLATE options. Valid options are 1, 2, and 4. Option 1 is fastest, while options 2 and 4 have progressively lower throughput but higher compression ratios. The default value is 1 when the user does not provide an input (i.e., None). | | Performance | `--gpusort` | No | Use GPUs to accelerate sorting and marking. | | Performance | `--use-gds` | No | Use GPUDirect Storage (GDS) to enable a direct data path for direct memory access (DMA) transfers between GPU memory and storage. Must be used concurrently with `--gpuwrite`. Please refer to Parabricks Documentation > Best Performance for information on how to set up and use GPUDirect Storage. | | Performance | `--memory-limit MEMORY_LIMIT` | No | System memory limit in GBs during sorting and postsorting. By default, the limit is 70% of the total system memory. | | Performance | `--low-memory` | No | Use low memory mode; will lower the number of streams per GPU. | | Runtime | `--verbose` | No | Enable verbose output. | | Runtime | `--x3` | No | Show full command line arguments. | | Runtime | `--dry-run` | No | Check if the current command is valid. | | Runtime | `--logfile LOGFILE` | No | Path to the log file. If not specified, messages will only be written to the standard error output. | | Runtime | `--tmp-dir TMP_DIR` | No | Full path to the directory where temporary files will be stored. (default: .) | | Runtime | `--with-petagene-dir WITH_PETAGENE_DIR` | No | Full path to the PetaGene installation directory. By default, this should have been installed at /opt/petagene. Use of this option also requires that the PetaLink library has been preloaded by setting the LD_PRELOAD environment variable. Optionally set the PETASUITE_REFPATH and PGCLOUD_CREDPATH environment variables that are used for data and credentials. Optionally set the PetaLinkMode environment variable that is used to further configure PetaLink, notably setting it to "+write" to enable outputting compressed BAM and .fastq files. | | Runtime | `--keep-tmp` | No | Do not delete the directory storing temporary files after completion. | | Runtime | `--no-seccomp-override` | No | Do not override seccomp options for docker. | | Runtime | `--version` | No | View compatible software versions. | | Runtime | `--preserve-file-symlinks` | No | Override default behavior to keep file symlinks intact and *not* resolve the symlink. | | Runtime | `--help-json` | No | Print out help message in JSON format. | | Runtime | `--num-gpus NUM_GPUS` | No | Number of GPUs to use for a run. (default: 1) | > **Note** > > The *--in-fq* option takes the names of two FASTQ files, optionally followed by a > quoted read group. The FASTQ filenames must not start with a hyphen. > **Note** > > When using the *--in-fq-list* option a read group is required on each line of the input file. [^bwameth-repo]: Baseline bwa-meth: [https://github.com/brentp/bwa-meth/](https://github.com/brentp/bwa-meth/) [^bwameth-manuscript]: Bwa-meth manuscript: [http://arxiv.org/abs/1401.1129](http://arxiv.org/abs/1401.1129) [^bwameth-doi]: [https://doi.org/10.1038/s41587-022-01336-9](https://doi.org/10.1038/s41587-022-01336-9)