Assembly (Long-read RNA)

Assemble transcripts from long-read RNA samples

tools

The following tools run by default (methods: "all"):

isonform, isonform_clustered, isonform-clustered, rattle, rattle_clustered, rattle-clustered, rnabloom2, rnabloom2_clustered, rnabloom2-clustered, stringtie3

usage

nexus run --nf-workflow assembly_long-read-rna.nf -params-file params.yaml

Note

Nextflow config files are available here. Use the config file that matches your installed nexus version (e.g. nexus_v0.2.0_nextflow_slurm.config).

parameters

Download params.yaml

# =============================================================================
# params.yaml — assembly_long-read-rna
#
# Usage:
#   nextflow run assembly_long-read-rna.nf -params-file params.yaml
#
# Fill in the required fields below. Optional fields can be left at their
# defaults or removed entirely.
# =============================================================================


# -----------------------------------------------------------------------------
# Required
# -----------------------------------------------------------------------------

# TSV file with columns:
#   sample_id
#   fastq_file      (raw long reads, for every method except stringtie3)
#   tsv_file        (per-read cluster assignments: 'cluster_id', 'read_name';
#                    for isonform_clustered, rattle_clustered, rnabloom2_clustered)
#   cluster_method  (name of the tool that produced tsv_file, e.g. 'isonclust3';
#                    required whenever a *_clustered method is active)
#   bam_file        (splice-aware aligned reads, e.g. minimap2, for stringtie3)
#   bam_bai_file    (index of bam_file, for stringtie3)
#
# To assemble one sample under SEVERAL clusterings, give it one row per
# clustering — repeat sample_id/fastq_file/bam_file and vary tsv_file +
# cluster_method:
#
#   sample_id  fastq_file  tsv_file        cluster_method  bam_file  bam_bai_file
#   rna-006    r.fq.gz     isonclust3.tsv  isonclust3      a.bam     a.bam.bai
#   rna-006    r.fq.gz     geluster.tsv    geluster        a.bam     a.bam.bai
#
# Clustered outputs are then written to, and named after, the clustering:
#   <output_dir>/rna-006/isonclust3_isonform_clustered/rna-006_isonclust3_isonform_merged.fasta.gz
#   <output_dir>/rna-006/geluster_isonform_clustered/rna-006_geluster_isonform_merged.fasta.gz
#
# (sample_id, cluster_method) must be unique — duplicates are rejected up front
# because they would overwrite each other. The de novo methods (isonform,
# rattle, rnabloom2) and stringtie3 ignore tsv_file/cluster_method and still run
# ONCE per sample no matter how many rows it has.
samples_tsv_file: ""

# Directory to which output files will be copied
output_dir: ""

# Reference genes annotation GTF file (reference-guided assembly)
# Required when methods includes 'stringtie3' or 'all'.
reference_genes_gtf_file: ""


# -----------------------------------------------------------------------------
# Optional — general
# -----------------------------------------------------------------------------

# Methods to run. Comma-separated list or 'all'.
# Allowed values:
#   all, isonform, isonform_clustered, rattle, rattle_clustered,
#   rnabloom2, rnabloom2_clustered, stringtie3
methods: "all"


# -----------------------------------------------------------------------------
# isONform (de novo, raw FASTQ)
# Optional arguments.
# -----------------------------------------------------------------------------
isonform:
  # Parameters passed directly to isON_pipeline.sh.
  extra_args: "--mode pacbio --iso_abundance 3"


# -----------------------------------------------------------------------------
# isONform (clustered; de novo per read cluster, raw FASTQ + cluster TSV)
# Optional arguments.
# -----------------------------------------------------------------------------
isonform_clustered:
  # Parameters passed directly to isONform_parallel. NOTE: these are
  # isONform_parallel's own flags, NOT isON_pipeline.sh's -- the clustering half
  # of that pipeline is replaced by the supplied cluster TSV, so there is no
  # --mode here. The default below reproduces isON_pipeline.sh's 'pacbio' mode.
  # Do not pass --write_fastq, or any of --t/--fastq_folder/--outfolder/
  # --tmpdir/--split_wrt_batches (set by the batch script).
  extra_args: "--k 20 --w 31 --xmin 14 --xmax 80 --exact_instance_limit 50 --max_seqs_to_spoa 200 --delta_len 10 --delta_iso_len_3 30 --delta_iso_len_5 50 --iso_abundance 3"


# -----------------------------------------------------------------------------
# RATTLE (de novo, raw FASTQ)
# Optional arguments.
# -----------------------------------------------------------------------------
rattle:
  # Parameters passed directly to `rattle cluster`.
  params_cluster: "--iso --rna"

  # Parameters passed directly to `rattle correct`.
  params_correct: ""

  # Parameters passed directly to `rattle polish`.
  params_polish: "--rna"


# -----------------------------------------------------------------------------
# RATTLE (clustered; de novo per read cluster, raw FASTQ + cluster TSV)
# The full cluster -> correct -> polish chain is run independently on each
# cluster. Do not pass --input/--clusters/--output/--output-folder/-t/--summary;
# the batch script sets those.
# Optional arguments.
# -----------------------------------------------------------------------------
rattle_clustered:
  # Parameters passed directly to `rattle cluster`.
  params_cluster: "--iso --rna"

  # Parameters passed directly to `rattle correct`.
  params_correct: ""

  # Parameters passed directly to `rattle polish`.
  params_polish: "--rna"


# -----------------------------------------------------------------------------
# RNA-Bloom2 (de novo, raw FASTQ)
# Optional arguments.
# -----------------------------------------------------------------------------
rnabloom2:
  # Parameters passed directly to RNA-Bloom2.
  extra_args: "--qual 20 --qual-avg 20 --mincov 3 -ntcard -savebf -chimera -lrpb"


# -----------------------------------------------------------------------------
# RNA-Bloom2 (clustered; de novo per read cluster, raw FASTQ + cluster TSV)
# Optional arguments.
# -----------------------------------------------------------------------------
rnabloom2_clustered:
  # Parameters passed directly to RNA-Bloom2.
  extra_args: "--qual 20 --qual-avg 20 --mincov 3 -ntcard -savebf -chimera -lrpb"


# -----------------------------------------------------------------------------
# StringTie3 (reference-guided, aligned BAM)
# Optional arguments.
# -----------------------------------------------------------------------------
stringtie3:
  # Parameters passed directly to StringTie3.
  extra_args: "-L"