Trinity

Introduction

The widely established tool for transcriptome assembly. It consists of three main components (which is how it got its name) although a fourth one, Jellyfish, is also included in the following list because it's important, thogh not written by the Trinity team:

• Jelly fish
• Inchworm
• Chrysalis
• Butterfly

This list follws the sequence of operations.

Version 2.3.2, full help file

This is an editted version. With the module loaded, type

Trinity --show-full-usage-info

For the raw version.

Obligatory options

• --seqType <string>, what type of reads the inputs are: ('fa' or 'fq')
• --max_memory <string>, suggested max memory to use by Trinity where limiting can be enabled. (jellyfish, sorting, etc) in Gb of RAM, ie. '--max_memory 10G'

• --left <string>  :left reads, one or more file names (separated by commas, no spaces)
• --right <string>  :right reads, one or more file names (separated by commas, no spaces)
• --single <string> (obviously neither obligatory nor necessary if paired reads are being input). Ssingle reads, one or more file names, comma-delimited (note, if single file contains pairs, can use flag: --run_as_paired )

Or,

• --samples_file <string> tab-delimited text file indicating biological replicate relationships.

1. ex.
2. cond_A cond_A_rep1 A_rep1_left.fq A_rep1_right.fq
3. cond_A cond_A_rep2 A_rep2_left.fq A_rep2_right.fq
4. cond_B cond_B_rep1 B_rep1_left.fq B_rep1_right.fq
5. cond_B cond_B_rep2 B_rep2_left.fq B_rep2_right.fq
7. # if single-end instead of paired-end, then leave the 4th column above empty.

Misc

• --SS_lib_type <string> Strand-specific RNA-Seq read orientation.

1. if paired: RF or FR,
2. if single: F or R. (dUTP method = RF)
3. See web documentation.
5. --CPU <int>  :number of CPUs to use, default: 2
6. --min_contig_length <int>  :minimum assembled contig length to report
7. (def=200)
9. --long_reads <string>  :fasta file containing error-corrected or circular consensus (CCS) pac bio reads
10. (** note: experimental parameter **, this functionality continues to be under development)
12. --genome_guided_bam <string>  :genome guided mode, provide path to coordinate-sorted bam file.
13. (see genome-guided param section under --show_full_usage_info)
15. --jaccard_clip  :option, set if you have paired reads and
16. you expect high gene density with UTR
17. overlap (use FASTQ input file format
18. for reads).
19. (note: jaccard_clip is an expensive
20. operation, so avoid using it unless
21. necessary due to finding excessive fusion
22. transcripts w/o it.)
24. --trimmomatic  :run Trimmomatic to quality trim reads
25. see '--quality_trimming_params' under full usage info for tailored settings.
28. --no_normalize_reads  :Do *not* run in silico normalization of reads. Defaults to max. read coverage of 50.
29. see '--normalize_max_read_cov' under full usage info for tailored settings.
30. (note, as of Sept 21, 2016, normalization is on by default)
32. --no_distributed_trinity_exec  :do not run Trinity phase 2 (assembly of partitioned reads), and stop after generating command list.
35. --output <string>  :name of directory for output (will be
36. created if it doesn't already exist)
37. default( your current working directory: "/storage/home/users/ramon/trinity_out_dir"
38. note: must include 'trinity' in the name as a safety precaution! )
40. --full_cleanup  :only retain the Trinity fasta file, rename as ${output_dir}.Trinity.fasta
42. --cite  :show the Trinity literature citation
44. --verbose  :provide additional job status info during the run.
46. --version  :reports Trinity version (Trinity-v2.3.2) and exits.

• -show_full_usage_info  :show the many many more options available for running Trinity (expert usage).

1. --KMER_SIZE <int>  :kmer length to use (default: 25) max=32
3. --prep  :Only prepare files (high I/O usage) and stop before kmer counting.
5. --no_cleanup  :retain all intermediate input files.
7. --no_version_check  :dont run a network check to determine if software updates are available.

Inchworm and K-mer counting-related options

• --min_kmer_cov <int>, min count for K-mers to be assembled by Inchworm (default: 1)
• --inchworm_cpu <int>, number of CPUs to use for Inchworm (only!), default is min(6, --CPU option)
• --no_run_inchworm, stop after running jellyfish, before inchworm. (phase 1, read clustering only)

Chrysalis-related options

1. --max_reads_per_graph <int>  :maximum number of reads to anchor within
2. a single graph (default: 200000)
3. --min_glue <int>  :min number of reads needed to glue two inchworm contigs
4. together. (default: 2)
6. --no_bowtie  :dont run bowtie to use pair info in chrysalis clustering.
8. --no_run_chrysalis  :stop after running inchworm, before chrysalis. (phase 1, read clustering only)

Butterfly-related options

• --bfly_opts <string>, additional parameters to pass through to butterfly see butterfly options:

java -jar Butterfly.jar

(note: only for expert or experimental use. Commonly used parameters are exposed through this Trinity menu here).

== Butterfly read-pair grouping settings (used to define 'pair paths'):

2. --group_pairs_distance <int>  :maximum length expected between fragment pairs (default: 500)
3. (reads outside this distance are treated as single-end)

1. Butterfly default reconstruction mode settings. (no CuffFly or PasaFly custom settings are currently available).
3. --path_reinforcement_distance <int>  :minimum overlap of reads with growing transcript
4. path (default: PE: 75, SE: 25)
5. Set to 1 for the most lenient path extension requirements.

1. Butterfly transcript reduction settings:
3. --no_path_merging  : all final transcript candidates are output (including SNP variations, however, some SNPs may be unphased)
5. By default, alternative transcript candidates are merged (in reality, discarded) if they are found to be too similar, according to the following logic:
7. (identity=(numberOfMatches/shorterLen) > 95.0% or if we have <= 2 mismatches) and if we have internal gap lengths <= 10
9. with parameters as:
11. --min_per_id_same_path <int> default: 98 min percent identity for two paths to be merged into single paths
12. --max_diffs_same_path <int> default: 2 max allowed differences encountered between path sequences to combine them
13. --max_internal_gap_same_path <int> default: 10 maximum number of internal consecutive gap characters allowed for paths to be merged into single paths.
15. If, in a comparison between two alternative transcripts, they are found too similar, the transcript with the greatest cumulative
16. compatible read (pair-path) support is retained, and the other is discarded.

Butterfly Java and parallel execution settings

• --bflyHeapSpaceMax <string>, java max heap space setting for butterfly (default: 4G this yields command

java -Xmx4G -jar Butterfly.jar ... $bfly_opts

• --bflyHeapSpaceInit <string>, java initial hap space settings for butterfly (default: 1G) this yields command

java -Xms1G -jar Butterfly.jar ... $bfly_opts

• --bflyGCThreads <int>, threads for garbage collection (default: 2))
• --bflyCPU <int>, CPUs to use (default will be normal number of CPUs; e.g., 2)
• --bflyCalculateCPU, Calculate CPUs based on 80% of max_memory divided by maxbflyHeapSpaceMax
• --bfly_jar <string>, /path/to/Butterfly.jar, otherwise default Trinity-installed version is used.

Quality Trimming Options

• --quality_trimming_params <string> defaults to: "ILLUMINACLIP:/usr/local/Modules/modulefiles/tools/trinity/2.3.2/trinity-plugins/Trimmomatic/adapters/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25"

In silico Read Normalization Options

2. --normalize_max_read_cov <int> defaults to 50
3. --normalize_by_read_set run normalization separate for each pair of fastq files,
4. then one final normalization that combines the individual normalized reads.
5. Consider using this if RAM limitations are a consideration.

Genome-guided de novo assembly

2. * required:
4. --genome_guided_max_intron <int>  :maximum allowed intron length (also maximum fragment span on genome)
6. * optional:
8. --genome_guided_min_coverage <int>  :minimum read coverage for identifying and expressed region of the genome. (default: 1)
10. --genome_guided_min_reads_per_partition <int>  :default min of 10 reads per partition

Trinity phase 2 (parallel assembly of read clusters) Options

• --grid_exec <string>, your command-line utility for submitting jobs to the grid. This should be a command line tool that accepts a single parameter:

${your_submission_tool} /path/to/file/containing/commands.txt and this submission tool should exit(0) upon successful                                     completion of all commands.

2. --grid_node_CPU <int> number of threads for each parallel process to leverage. (default: 1)
4. --grid_node_max_memory <string> max memory targeted for each grid node. (default: 1G)
6. The --grid_node_CPU and --grid_node_max_memory are applied as
7. the --CPU and --max_memory parameters for the Trinity jobs run in
8. Trinity Phase 2 (assembly of read clusters)

A typical Trinity command might be

Trinity --seqType fq --max_memory 50G --left reads_1.fq  --right reads_2.fq --CPU 6

and for Genome-guided Trinity:

Trinity --genome_guided_bam rnaseq_alignments.csorted.bam --max_memory 50G --genome_guided_max_intron 10000 --CPU 6

• see:

/usr/local/Modules/modulefiles/tools/trinity/2.3.2/sample_data/test_Trinity_Assembly/ for sample data and 'runMe.sh' for example Trinity execution