labxpipe 0.4.0

Genomics pipelines

• Integrated with LabxDB: all required annotations (labels, strand, paired etc) are retrieved from LabxDB. This is optional.
• Based on existing robust technologies. No new language.
  • LabxPipe pipelines are defined in JSON text files.
  • LabxPipe is written in Python. Using norms, such as input and output filenames, insures compatibility between tasks.
• Simple and complex pipelines.
  • By default, pipelines are linear (one step after the other).
  • Branching is easily achieved be defining a previous step (using step_input parameter) allowing users to create any dependency between tasks.
• Parallelized using robust asynchronous threads from the Python standard library.

Examples

See JSON files in config/pipelines of this repository.

Pipeline JSON file
mrna_seq.json	mRNA-seq
mrna_seq_no_db.json	mRNA-seq. No LabxDB
mrna_seq_with_plotting.json	mRNA-seq. Plotting non-mapped reads. Demonstrate step_input
mrna_seq_cufflinks.json	mRNA-seq. Replaces GeneAbacus by Cufflinks
chip_seq.json	ChIP-seq using Bowtie2 and Samtools to uniquify reads.

Following demonstrates how to apply mrna_seq.json pipeline. It requires:

• LabxDB
• FASTQ files for sample named AGR000850 and AGR000912

  /plus/data/seq/by_run/AGR000850
  ├── 23_009_R1.fastq.zst
  └── 23_009_R2.fastq.zst
  /plus/data/seq/by_run/AGR000912
  ├── 65_009_R1.fastq.zst
  └── 65_009_R2.fastq.zst
  

Note: mrna_seq_no_db.json demonstrates how to use LabxPipe without LabxDB: it only requires FASTQ files (in path_seq_run directory, see above).

Requirements:

• LabxDB. Alternatively, mrna_seq_no_db.json doesn't require LabxDB.
• ReadKnead to trim reads.
• STAR and genome index in directory defined path_star_index.
• GeneAbacus to count reads and generate genomic profile for tracks.

1. Start pipeline:

   lxpipe run --pipeline mrna_seq.json \
              --worker 2 \
              --processor 16
   

   Output is written in path_output directory.

2. Create report:

   lxpipe report --pipeline mrna_seq.json
   

   Report file mrna_seq.xlsx should be created in same directory as mrna_seq.json.

3. Merge gene/mRNA counts generated by GeneAbacus in counting directory:

   lxpipe merge-count --pipeline mrna_seq.json \
                      --step counting
   

4. Trackhub. Requirements:

   • ChromosomeMappings file (to map chromosome names from Ensembl/NCBI to UCSC)
   • Tabulated file (with chromosome name and length)

   Execute in a separate directory:

   lxpipe trackhub --runs AGR000850,AGR000912 \
                   --species_ucsc danRer11 \
                   --path_genome /plus/scratch/sai/annots/danrer_genome_all_ensembl_grcz11_ucsc_chroms_chrom_length.tab \
                   --path_mapping /plus/scratch/sai/annots/ChromosomeMappings/GRCz11_ensembl2UCSC.txt \
                   --input_sam \
                   --bam_names accepted_hits.sam.zst \
                   --make_config \
                   --make_trackhub \
                   --make_bigwig \
                   --processor 16
   

   Directory is ready to be shared by a web server for display in the UCSC genome browser.

Configuration

Parameters can be defined globally. See in config directory of this repository for examples.

Writing pipelines

Parameters are defined first globally (see above), then per pipeline, then per replicate/run, and then per step/function. The latest definition takes precedence: path_seq_run defined in /etc/hts/labxpipe.json is used by default, but if path_seq_run is defined in the pipeline file, it will be used instead.

Main parameters

Parameter	Type
name	string
path_output	string
path_seq_run	string
path_annots	string
path_bowtie2_index	string
path_star_index	string
fastq_exts	[]strings
adaptors	{}
logging_level	string
run_refs	[]strings
replicate_refs	[]strings
ref_info_source	[]strings
ref_infos	{}
analysis	[{}, {}, ...]

Parameters for all functions

Parameter	Type
step_name	string
step_function	string
step_desc	string
force	boolean

Function-specific parameters

Function	Synonym	Parameter	Type
readknead	preparing	options	[]strings
		ops_r1	[{}, {}, ...]
		ops_r2	[{}, {}, ...]
		plot_fastq_in	boolean
		plot_fastq	boolean
		fastq_out	boolean
		zip_fastq_out	string
bowtie2	genomic_aligning	options	[]strings
		index	string
		output	string
		output_unfiltered	string
		compress_sam	boolean
		compress_sam_cmd	string
		create_bam	boolean
		index_bam	boolean
star	aligning	options	[]strings
		index	string
		output_type	[]strings
		compress_sam	boolean
		compress_sam_cmd	string
		compress_unmapped	boolean
		compress_unmapped_cmd	string
cufflinks		options	[]strings
		inputs	[{}, {}, ...]
		features	[{}, {}, ...]
geneabacus	counting	options	[]strings
		inputs	[{}, {}, ...]
		features	[{}, {}, ...]
uniquify		options	[]strings
		sort_by_name_bam	boolean
		index_bam	boolean
cleaning		steps	[{}, {}, ...]

Sample-specific parameters. Automatically populated if using LabxDB or sourced from ref_infos. These parameters can be changed manually in any function (for example setting paired to False will ignore second reads in that step).

Parameter	Type
label_short	string
paired	boolean
directional	boolean
r1_strand	string
quality_scores	string

Demultiplexing sequencing reads: lxpipe demultiplex

• Demultiplex reads based on barcode sequences from the Second barcode field in LabxDB

• Demultiplexing using ReadKnead. The most important for demultiplexing is the ReadKnead pipeline. Pipelines are identified using the Adapter 3' field in LabxDB.

• Example for simple demultiplexing. The first nucleotides at the 5' end of read 1 are used as barcodes (the Adapter 3' field is set to sRNA 1.5 in LabxDB for these samples) with the following pipeline:

  {
      "sRNA 1.5": {
          "R1": [{"name": "demultiplex",
                  "end": 5,
                  "max_mismatch": 1}],
          "R2": null
      }
  }
  

  The barcode sequences are added by LabxPipe using the Second barcode field in LabxDB.

• Example for iCLIP demultiplexing. In Vejnar et al., iCLIP is demultiplexed (the Adapter 3' field is set to TruSeq-DMS+A Index in LabxDB for these samples) using the following pipeline:

  {
      "TruSeq-DMS+A Index": {
          "R1": [{"name": "clip",
                  "end": 5,
                  "length": 4,
                  "add_clipped": true},
              {"name": "trim",
               "end": 3,
               "algo": "bktrim",
               "min_sequence": 5,
               "keep": ["trim_exact", "trim_align"]},
              {"name": "length",
               "min_length": 6},
              {"name": "demultiplex",
               "end": 3,
               "max_mismatch": 1,
               "length_ligand": 2},
              {"name": "length",
               "min_length": 15}],
          "R2": null
      }
  }
  

  Pipeline is stored in demux_truseq_dms_a.json. The barcode sequences are added by LabxPipe using the Second barcode field in LabxDB. (NB: published demultiplexed data were generated using "algo": "align" with a minimum score of 80 instead of "algo": "bktrim")

  Then pipeline was tested running:

  lxpipe demultiplex --bulk HHYLKADXX \
                     --path_demux_ops demux_truseq_dms_a.json \
                     --path_seq_prepared prepared \
                     --demux_nozip \
                     --processor 1 \
                     --demux_verbose_level 20 \
                     --no_readonly
  

  This output is very verbose: for every read, output from every step of the demultiplexing pipeline is reported. To get consistent output, --processor must be set to 1. Output is written in local directory prepared.

  And finally, once pipeline is validated (data is written in path_seq_prepared directory, see here):

  lxpipe demultiplex --bulk HHYLKADXX \
                     --path_demux_ops demux_truseq_dms_a.json \
                     --processor 10
  

License

LabxPipe is distributed under the Mozilla Public License Version 2.0 (see /LICENSE).

Copyright © 2013-2023 Charles E. Vejnar