Trinityはバグ修正と性能改善のバージョンアップが続けられていて、2022年5月現在ではv2.14が利用できます。v2.14はまだcondaでは導入できないので、ソースからビルドします。
Installing Trinity
https://github.com/trinityrnaseq/trinityrnaseq/wiki/Installing-Trinity
インストール
依存
- bowtie2
- jellyfish
- salmon
- samtools
mamba create -n trinity python=3.9
conda activate trinity
mamba install -c bioconda -y kmer-jellyfish #もしくはソースからビルドする(参考)
mamba install -c bioconda salmon -y
mamba install -c bioconda bowtie2=2.4.5 -y #少し古いバージョンだとエラーになる。
mamba install -c bioconda samtools -y #もしくはソースからビルドする(参考)
#本体(2022年12月に2.15が出た、2023年2月現在v2.15.1が最新)
wget https://github.com/trinityrnaseq/trinityrnaseq/releases/download/Trinity-v2.15.0/trinityrnaseq-v2.15.0.FULL.tar.gz
tar -xvf trinityrnaseq-v2.15.0.FULL.tar.gz
cd trinityrnaseq-v2.15.0/
make -j20
make plugins # build the additional plugin components
sudo make install
#perl dbのエラーが出るなら
mamba install -c bioconda perl-db-file -y
#trinity本体はcondaでも導入できる(conda配布版は2.13 2.15.1が最新(上のリンクから確認)
#bioconda(link)
mamba create -n trinity python=3.9
conda activate trinity
#バージョン指定しないと古いバージョンが入ることがある。バージョンを指定する。
mamba install -c conda-forge -c bioconda -y trinity=2.15.1
> Trinity
###############################################################################
#
______ ____ ____ ____ ____ ______ __ __
| || \ | || \ | || || | |
| || D ) | | | _ | | | | || | |
|_| |_|| / | | | | | | | |_| |_|| ~ |
| | | \ | | | | | | | | | |___, |
| | | . \ | | | | | | | | | | |
|__| |__|\_||____||__|__||____| |__| |____/
Trinity-v2.14.0
#
#
# Required:
#
# --seqType <string> :type of reads: ('fa' or 'fq')
#
# --max_memory <string> :suggested max memory to use by Trinity where limiting can be enabled. (jellyfish, sorting, etc)
# provided in Gb of RAM, ie. '--max_memory 10G'
#
# If paired reads:
# --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)
#
# Or, if unpaired reads:
# --single <string> :single 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.
# ex.
# cond_A cond_A_rep1 A_rep1_left.fq A_rep1_right.fq
# cond_A cond_A_rep2 A_rep2_left.fq A_rep2_right.fq
# cond_B cond_B_rep1 B_rep1_left.fq B_rep1_right.fq
# cond_B cond_B_rep2 B_rep2_left.fq B_rep2_right.fq
#
# # 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.
# if paired: RF or FR,
# if single: F or R. (dUTP method = RF)
# See web documentation.
#
# --CPU <int> :number of CPUs to use, default: 2
# --min_contig_length <int> :minimum assembled contig length to report
# (def=200, must be >= 100)
#
# --long_reads <string> :fasta file containing error-corrected or circular consensus (CCS) pac bio reads
# (** note: experimental parameter **, this functionality continues to be under development)
#
# --genome_guided_bam <string> :genome guided mode, provide path to coordinate-sorted bam file.
# (see genome-guided param section under --show_full_usage_info)
#
# --long_reads_bam <string> :long reads to include for genome-guided Trinity
# (bam file consists of error-corrected or circular consensus (CCS) pac bio read aligned to the genome)
#
# --jaccard_clip :option, set if you have paired reads and
# you expect high gene density with UTR
# overlap (use FASTQ input file format
# for reads).
# (note: jaccard_clip is an expensive
# operation, so avoid using it unless
# necessary due to finding excessive fusion
# transcripts w/o it.)
#
# --trimmomatic :run Trimmomatic to quality trim reads
# see '--quality_trimming_params' under full usage info for tailored settings.
#
# --output <string> :name of directory for output (will be
# created if it doesn't already exist)
# default( your current working directory: "/media/kazu/4TB4/Sugimoto_denovoRNAseq/trinity_out_dir"
# note: must include 'trinity' in the name as a safety precaution! )
#
# --full_cleanup :only retain the Trinity fasta file, rename as ${output_dir}.Trinity.fasta
#
# --cite :show the Trinity literature citation
#
# --verbose :provide additional job status info during the run.
#
# --version :reports Trinity version (Trinity-v2.14.0) and exits.
#
# --show_full_usage_info :show the many many more options available for running Trinity (expert usage).
#
#
###############################################################################
#
# *Note, a typical Trinity command might be:
#
# Trinity --seqType fq --max_memory 50G --left reads_1.fq --right reads_2.fq --CPU 6
#
# (if you have multiple samples, use --samples_file ... see above for details)
#
# and for Genome-guided Trinity, provide a coordinate-sorted bam:
#
# Trinity --genome_guided_bam rnaseq_alignments.csorted.bam --max_memory 50G
# --genome_guided_max_intron 10000 --CPU 6
#
# see: /home/kazu/Documents/trinityrnaseq-v2.14.0.FULL/trinityrnaseq-v2.14.0/sample_data/test_Trinity_Assembly/
# for sample data and 'runMe.sh' for example Trinity execution
#
# For more details, visit: http://trinityrnaseq.github.io
#
###############################################################################
導入できました。v2.14ではデフォルトの最小コンティグ長が100-ntになっているので、今までのバージョンと比較する時は注意して下さい。
ラン例
De novo transcriptome assembly
#全サンプルをmergeしたfastqを使い、de novoでアセンブル
Trinity --seqType fq --left R1.fq.gz --right R2.fq.gz --max_memory 200G --CPU 40 --output Trinity_outdir
-
--SS_lib_type Strand-specific RNA-Seq read orientation. if paired: RF or FR, if single: F or R. (dUTP method = RF) See web documentation.
-
--left left reads, one or more file names (separated by commas, no spaces)
- --right right reads, one or more file names (separated by commas, no spaces)
- --single single reads, one or more file names, comma-delimited (note, if single file contains pairs, can use flag: --run_as_paired )
-
--samples_file tab-delimited text file indicating biological replicate relationships.
- --CPU number of CPUs to use, default: 2
-
--long_reads asta file containing error-corrected or circular consensus (CCS) pac bio reads (** note: experimental parameter **, this functionality continues to be under development)
-
--genome_guided_bam genome guided mode, provide path to coordinate-sorted bam file.
-
--output name of directory for output (will be created if it doesn't already exist)
- --full_cleanup only retain the Trinity fasta file, rename as ${output_dir}.Trinity.fasta
- --jaccard_clip option, set if you have paired reads and you expect high gene density with UTR overlap (use FASTQ input file format for reads). (note: jaccard_clip is an expensive operation, so avoid using it unless necessary due to finding excessive fusion transcripts w/o it.) (参考)
しばらく前のバージョンからcDNAロングリードを使ったアセンブリもできるようになっていますね。機会があれば試してみたいです。
引用
Full-length transcriptome assembly from RNA-Seq data without a reference genome.
Manfred G Grabherr, Brian J Haas, Moran Yassour, Joshua Z Levin, Dawn A Thompson, Ido Amit, Xian Adiconis, Lin Fan, Raktima Raychowdhury, Qiandong Zeng, Zehua Chen, Evan Mauceli, Nir Hacohen, Andreas Gnirke, Nicholas Rhind, Federica di Palma, Bruce W Birren, Chad Nusbaum, Kerstin Lindblad-Toh, Nir Friedman & Aviv Regev
Nature Biotechnology 29, 644–652 (2011)
関連