rna_fq2bam
Spliced Transcripts Alignment to a Reference.
$ pbrun rna_fq2bam \
--in-fq sample_X_1.fq.gz sample_X_2.fq.gz \
--genome-lib-dir HG38 \
--output-dir sample_X/ \
--ref ref.fasta
The output from these commands will be identical to the output from the above command. See the Output Comparison page for comparing the results.
#STAR Alignment
$ ./STAR --genomeDir HG38 --readFilesIn sample_X_1.fq.gz sample_X_2.fq.gz
--outFileNamePrefix sample_X/ --outSAMtype BAM SortedByCoordinate
#Coordinate Sorting
$ gatk SortSam --java-options -Xmx30g --MAX_RECORDS_IN_RAM=5000000 -I=Aligned.sortedByCoord.out.bam \
-O=cpu.bam --SORT_ORDER=coordinate --TMP_DIR=/raid/myrun
# Mark Duplicates
$ gatk MarkDuplicates --java-options -Xmx30g -I=cpu.bam -O=mark_dups_cpu.bam \
-M=metrics.txt --TMP_DIR=/raid/myrun
Run RNA-seq data through the fq2bam pipeline. It will run STAR aligner, co-ordinate sorting and mark duplicates.
Input/Output file options
- --ref REF
- --in-fq [IN_FQ [IN_FQ ...]]
- --in-se-fq [IN_SE_FQ [IN_SE_FQ ...]]
- --genome-lib-dir GENOME_LIB_DIR
- --output-dir OUTPUT_DIR
- --out-bam OUT_BAM
Path to the reference file. (default: None)
Option is required.
Path to the pair ended FASTQ files followed by optional read group with quotes (Example: "@RGtID:footLB:lib1tPL:bartSM:sampletPU:foo"). Files must be in fastq/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 "@RGtID:footLB:lib1tPL:bartSM:sampletPU:unit1" --in-fq sampleX_2_1.fastq.gz sampleX_2_2.fastq.gz "@RGtID:foo2tLB:lib1tPL:bartSM:sampletPU:unit2" . For same sample, Read Groups should have same sample name (SM) and different ID and PU (default: None)
Path to the single ended FASTQ file followed by optional read group with quotes (Example: "@RGtID:footLB:lib1tPL:bartSM:sampletPU:foo"). The file must be in fastq/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-se-fq sampleX_1.fastq.gz --in-se-fq sampleX_2.fastq.gz . Example 2: --in-se-fq sampleX_1.fastq.gz "@RGtID:footLB:lib1tPL:bartSM:sampletPU:unit1" --in-se-fq sampleX_2.fastq.gz "@RGtID:foo2tLB:lib1tPL:bartSM:sampletPU:unit2" . For same sample, Read Groups should have same sample name (SM) and different ID and PU (default: None)
Path to a genome resource library directory. We assume that the indexing required to run star has been completed by the user. (default: None)
Option is required.
Path to the directory that will contain all of the generated files. (default: None)
Option is required.
Path of output BAM file. (default: None)
Option is required.
Options specific to this tool
- --num-threads NUM_THREADS
- --out-prefix OUT_PREFIX
- --read-files-command READ_FILES_COMMAND
- --read-group-sm READ_GROUP_SM
- --read-group-lb READ_GROUP_LB
- --read-group-pl READ_GROUP_PL
- --read-group-id-prefix READ_GROUP_ID_PREFIX
- --num-sa-bases NUM_SA_BASES
- --max-intron-size MAX_INTRON_SIZE
- --min-intron-size MIN_INTRON_SIZE
- --min-match-filter MIN_MATCH_FILTER
- --min-match-filter-normalized MIN_MATCH_FILTER_NORMALIZED
- --out-filter-intron-motifs OUT_FILTER_INTRON_MOTIFS
- --max-out-filter-mismatch MAX_OUT_FILTER_MISMATCH
- --max-out-filter-mismatch-ratio MAX_OUT_FILTER_MISMATCH_RATIO
- --max-out-filter-multimap MAX_OUT_FILTER_MULTIMAP
- --out-reads-unmapped OUT_READS_UNMAPPED
- --out-sam-unmapped OUT_SAM_UNMAPPED
- --out-sam-attributes OUT_SAM_ATTRIBUTES [OUT_SAM_ATTRIBUTES ...]
- --out-sam-strand-field OUT_SAM_STRAND_FIELD
- --out-sam-mode OUT_SAM_MODE
- --out-sam-mapq-unique OUT_SAM_MAPQ_UNIQUE
- --min-score-filter MIN_SCORE_FILTER
- --min-spliced-mate-length MIN_SPLICED_MATE_LENGTH
- --max-junction-mismatches MAX_JUNCTION_MISMATCHES MAX_JUNCTION_MISMATCHES MAX_JUNCTION_MISMATCHES MAX_JUNCTION_MISMATCHES
- --max-out-read-size MAX_OUT_READ_SIZE
- --max-alignments-per-read MAX_ALIGNMENTS_PER_READ
- --score-gap SCORE_GAP
- --seed-search-start SEED_SEARCH_START
- --max-bam-sort-memory MAX_BAM_SORT_MEMORY
- --align-ends-type ALIGN_ENDS_TYPE
- --align-insertion-flush ALIGN_INSERTION_FLUSH
- --max-align-mates-gap MAX_ALIGN_MATES_GAP
- --min-align-spliced-mate-map MIN_ALIGN_SPLICED_MATE_MAP
- --max-collapsed-junctions MAX_COLLAPSED_JUNCTIONS
- --min-align-sj-overhang MIN_ALIGN_SJ_OVERHANG
- --min-align-sjdb-overhang MIN_ALIGN_SJDB_OVERHANG
- --sjdb-overhang SJDB_OVERHANG
- --min-chim-overhang MIN_CHIM_OVERHANG
- --min-chim-segment MIN_CHIM_SEGMENT
- --max-chim-multimap MAX_CHIM_MULTIMAP
- --chim-multimap-score-range CHIM_MULTIMAP_SCORE_RANGE
- --chim-score-non-gtag CHIM_SCORE_NON_GTAG
- --min-non-chim-score-drop MIN_NON_CHIM_SCORE_DROP
- --out-chim-format OUT_CHIM_FORMAT
- --two-pass-mode TWO_PASS_MODE
- --out-chim-type OUT_CHIM_TYPE
- --read-name-separator READ_NAME_SEPARATOR [READ_NAME_SEPARATOR ...]
Number of running worker threads per GPU. (default: 4)
Prefix filename for output data. (default: None)
Command line to execute for each of the input files. This command should generate FASTA or FASTQ text and send it to stdout. For example: zcat - to uncompress .gz files, bzcat - to uncompress .bz2 files, etc. (default: None)
SM tag for read groups in this run. (default: None)
LB tag for read groups in this run. (default: None)
PL tag for read groups in this run. (default: None)
prefix for ID and PU tag for read groups in this run. This prefix will be used for all pair of FASTQ files in this run. The ID and PU tag will consist of this prefix and an identifier which will be unique for a pair of FASTQ files. (default: None)
Length (bases) of the SA pre-indexing string. Longer strings will use much more memory, but allow for faster searches. A value between 10 and 15 is recommended. For small genomes, the parameter must be scaled down to min(14, log2(GenomeLength)/2 - 1). (default: 14)
Maximum align intron size. If this value is 0, the maximum size will be determined by (2^winBinNbits)*winAnchorDistNbins. (default: 0)
Minimum align intron size. Genomic gap is considered intron if its length is greater than or equal to this value, otherwise it is considered Deletion. (default: 21)
Minimum number of matched bases required for alignment output. (default: 0)
Same as --min-match-filter, but normalized to the read length (sum of mates' lengths for paired-end reads). (default: 0.66)
Type of filter alignment using their motifs. This string can be "None" for no filtering, "RemoveNoncanonical" for filtering out alignments that contain non-canonical junctions, or "RemoveNoncanonicalUnannotated" for filtering out alignments that contain non-canonical unannotated junctions when using annotated splice junctions database. The annotated non-canonical junctions will be kept. (default: None)
Maximum number of mismatches allowed for an alignment to be output. (default: 10)
Maximum ratio of mismatches to mapped length allowed for an alignment to be output. (default: 0.3)
Maximum number of loci the read is allowed to map to for all alignments to be output. Otherwise, no alignments will be output and the read will be counted as "mapped to too many loci" in the Log.final.out. (default: 10)
Type of output of unmapped and partially mapped (i.e. mapped only one mate of a paired end read) reads in separate file(s). This string can be "None" for no output or "Fastx" for output in separate FASTA/FASTQ files, Unmapped.out.mate1/2. (default: None)
Type of output of unmapped reads in the SAM format. The string can be "None" for no output, "Within" to output unmapped reads within the main SAM file, "KeepPairs" for no output and unmapped mates will be recorded for each alignment, or "Within_KeepPairs" to output unmapped reads within the main SAM file and unmapped mates will be recorded for each alignment. (default: None)
A string of SAM attributes in the order desired for the output SAM. The string can contain any combination of the following attributes: {NH, HI, AS, nM, NM, MD, jM, jI, XS, MC, ch}. Alternatively, the string can be "None" for no attributes, "Standard" for the attributes {NH, HI, AS, nM}, or "All" for the attributes {NH, HI, AS, nM, NM, MD, jM, jI, MC, ch}. e.g. '--outSAMattributes NH nM jI XS ch'. (default: Standard)
Cufflinks-like strand field flag. The string can be "None" for no flag or "intronMotif" for the strand derived from the intron motif. Reads with inconsistent and/or non-canonical introns will be filtered out. (default: None)
SAM output mode. The string can be "None" for no SAM output, "Full" for full SAM output, or "NoQS" for full SAM output without quality scores. (default: Full)
The MAPQ value for unique mappers. Must be in the range [0, 255]. (default: 255)
Minimum score required for alignment output, normalized to the read length (sum of mates' lengths for paired-end reads). (default: 0.66)
Minimum mapped length for a read mate that is spliced, normalized to the mate length. Must be greater than 0. (default: 0.66)
Maximum number of mismatches for stitching of the splice junctions. A limit must be specified for each of the following: (1) non-canonical motifs, (2) GT/AG and CT/AC motif, (3) GC/AG and CT/GC motif, (4) AT/AC and GT/AT motif. To indicate no limit for any of the four options, use -1. (default: [0, -1, 0, 0])
Maximum size of the SAM record (bytes) for one read. Recommended value: >(2*(LengthMate1+LengthMate2+100)*o utFilterMultimapNmax. Must be greater than 0. (default: 100000)
Maximum number of different alignments per read to consider. Must be greater than 0. (default: 10000)
Splice junction penalty (independent on intron motif). (default: 0)
Defines the search start point through the read. The read split pieces will not be longer than this value. Must be greater than 0. (default: 50)
Maximum available RAM (bytes) for sorting BAM. If this value is 0, it will be set to the genome index size. Must be greater than or equal to 0. (default: 0)
Type of read ends alignment. Can be one of two options: "Local" will do a standard local alignment with soft-clipping allowed; "EndToEnd" will force an end-to-end read alignment with no soft-clipping. (default: Local)
How to flush ambiguous insertion positions. The string can be "None" to not flush insertions or "Right" to flush insertions to the right. (default: None)
Maximum gap between two mates. If 0, max intron gap will be determined by (2^winBinNbits)*winAnchorDistNbins. (default: 0)
Minimum mapped length for a read mate that is spliced. Must be greater than or equal to 0. (default: 0)
Maximum number of collapsed junctions. Must be greater than 0. (default: 1000000)
Minimum overhang (i.e. block size) for spliced alignments. Must be greater than 0. (default: 5)
Minimum overhang (i.e. block size) for annotated (sjdb) spliced alignments. Must be greater than 0. (default: 3)
Length of the donor/acceptor sequence on each side of the junctions. Ideally this value should be equal to mate_length - 1. Must be greater than 0. (default: 100)
Minimum overhang for the Chimeric.out.junction file. Must be greater than or equal to 0. (default: 20)
Minimum chimeric segment length. If it is set to 0, there will be no chimeric output. Must be greater than or equal to 0. (default: 0)
Maximum number of chimeric multi-alignments. If it is set to 0, the old scheme for chimeric detection which only considered unique alignments will be used. Must be greater than or equal to 0. (default: 0)
The score range for multi-mapping chimeras below the best chimeric score. This option only works with --max-chim-multimap > 1. Must be greater than or equal to 0. (default: 1)
The penalty for a non-GT/AG chimeric junction. (default: -1)
To trigger chimeric detection, the drop in the best non-chimeric alignment score with respect to the read length has to be smaller than this value. Must be greater than or equal to 0. (default: 20)
Formatting type for the Chimeric.out.junction file. Possible types are {0, 1}. If type 0, there will be no comment lines/headers. If type 1, there will be comment lines at the end of the file: command line and Nreads: total, unique, multi. (default: 0)
2-pass mapping mode. The string can be "None" for 1-pass mapping or "Basic" for basic 2-pass mapping with all 1st pass junctions inserted into the genome indices on the fly. (default: None)
Type of chimeric output. This string can be "Junctions" for Chimeric.out.junction, "WithinBAM" for main aligned BAM files (Aligned.*.bam), "WithinBAM_HardClip" for hard-clipping in the CIGAR for supplemental chimeric alignments, or "WithinBAM_SoftClip" for soft-clipping in the CIGAR for supplemental chimeric alignments. (default: Junctions)
Character(s) separating the part of the read names that will be trimmed in output (read name after space is always trimmed). (default: /)
Common options:
- --logfile LOGFILE
- --tmp-dir TMP_DIR
- --with-petagene-dir WITH_PETAGENE_DIR
- --keep-tmp
- --license-file LICENSE_FILE
- --no-seccomp-override
- --version
Path to the log file. If not specified, messages will only be written to the standard error output. (default: None)
Full path to the directory where temporary files will be stored.
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 (default: None)
Do not delete the directory storing temporary files after completion.
Path to license file license.bin if not in the installation directory.
Do not override seccomp options for docker (default: None).
View compatible software versions.
GPU options:
- --num-gpus NUM_GPUS
- --gpu-devices GPU_DEVICES
Number of GPUs to use for a run. GPUs 0..(NUM_GPUS-1) will be used.
GPU devices to use for a run. By default, all GPU devices will be used.
To use specific GPU devices, enter a comma-separated list of GPU device
numbers. Possible device numbers can be found by examining the output of
the nvidia-smi command. For example, using --gpu-devices 0,1
would only use the first two GPUs.
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.