Left read files: $VAR1 = [ '/gscratch/srlab/sr320/data/crab/304428_S1_L001_R1_001.fastq.gz', '/gscratch/srlab/sr320/data/crab/304428_S1_L002_R1_001.fastq.gz' ]; Right read files: $VAR1 = [ '/gscratch/srlab/sr320/data/crab/304428_S1_L001_R2_001.fastq.gz', '/gscratch/srlab/sr320/data/crab/304428_S1_L002_R2_001.fastq.gz' ]; Trinity version: Trinity-v2.4.0 -ERROR: couldn't run the network check to confirm latest Trinity software version. Friday, October 26, 2018: 06:25:38 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /gscratch/srlab/programs/trinity/util/support_scripts/ExitTester.jar 0 Friday, October 26, 2018: 06:25:38 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /gscratch/srlab/programs/trinity/util/support_scripts/ExitTester.jar 1 ---------------------------------------------------------------------------------- -------------- Trinity Phase 1: Clustering of RNA-Seq Reads --------------------- ---------------------------------------------------------------------------------- --------------------------------------------------------------- ------ Quality Trimming Via Trimmomatic --------------------- << ILLUMINACLIP:/gscratch/srlab/programs/trinity/trinity-plugins/Trimmomatic/adapters/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25 >> --------------------------------------------------------------- ## Running Trimmomatic on read files: /gscratch/srlab/sr320/data/crab/304428_S1_L001_R1_001.fastq.gz, /gscratch/srlab/sr320/data/crab/304428_S1_L001_R2_001.fastq.gz Friday, October 26, 2018: 06:25:39 CMD: java -jar /gscratch/srlab/programs/trinity/trinity-plugins/Trimmomatic/trimmomatic.jar PE -threads 28 -phred33 /gscratch/srlab/sr320/data/crab/304428_S1_L001_R1_001.fastq.gz /gscratch/srlab/sr320/data/crab/304428_S1_L001_R2_001.fastq.gz 304428_S1_L001_R1_001.fastq.gz.P.qtrim 304428_S1_L001_R1_001.fastq.gz.U.qtrim 304428_S1_L001_R2_001.fastq.gz.P.qtrim 304428_S1_L001_R2_001.fastq.gz.U.qtrim ILLUMINACLIP:/gscratch/srlab/programs/trinity/trinity-plugins/Trimmomatic/adapters/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25 TrimmomaticPE: Started with arguments: -threads 28 -phred33 /gscratch/srlab/sr320/data/crab/304428_S1_L001_R1_001.fastq.gz /gscratch/srlab/sr320/data/crab/304428_S1_L001_R2_001.fastq.gz 304428_S1_L001_R1_001.fastq.gz.P.qtrim 304428_S1_L001_R1_001.fastq.gz.U.qtrim 304428_S1_L001_R2_001.fastq.gz.P.qtrim 304428_S1_L001_R2_001.fastq.gz.U.qtrim ILLUMINACLIP:/gscratch/srlab/programs/trinity/trinity-plugins/Trimmomatic/adapters/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25 Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT' ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences Input Read Pairs: 31231444 Both Surviving: 30364672 (97.22%) Forward Only Surviving: 863407 (2.76%) Reverse Only Surviving: 0 (0.00%) Dropped: 3365 (0.01%) TrimmomaticPE: Completed successfully Friday, October 26, 2018: 06:26:43 CMD: cp 304428_S1_L001_R1_001.fastq.gz.P.qtrim 304428_S1_L001_R1_001.fastq.gz.PwU.qtrim.fq Friday, October 26, 2018: 06:26:49 CMD: cp 304428_S1_L001_R2_001.fastq.gz.P.qtrim 304428_S1_L001_R2_001.fastq.gz.PwU.qtrim.fq Friday, October 26, 2018: 06:26:56 CMD: touch trimmomatic.ok Friday, October 26, 2018: 06:26:56 CMD: gzip 304428_S1_L001_R1_001.fastq.gz.P.qtrim 304428_S1_L001_R1_001.fastq.gz.U.qtrim 304428_S1_L001_R2_001.fastq.gz.P.qtrim 304428_S1_L001_R2_001.fastq.gz.U.qtrim & ## Running Trimmomatic on read files: /gscratch/srlab/sr320/data/crab/304428_S1_L002_R1_001.fastq.gz, /gscratch/srlab/sr320/data/crab/304428_S1_L002_R2_001.fastq.gz Friday, October 26, 2018: 06:26:56 CMD: java -jar /gscratch/srlab/programs/trinity/trinity-plugins/Trimmomatic/trimmomatic.jar PE -threads 28 -phred33 /gscratch/srlab/sr320/data/crab/304428_S1_L002_R1_001.fastq.gz /gscratch/srlab/sr320/data/crab/304428_S1_L002_R2_001.fastq.gz 304428_S1_L002_R1_001.fastq.gz.P.qtrim 304428_S1_L002_R1_001.fastq.gz.U.qtrim 304428_S1_L002_R2_001.fastq.gz.P.qtrim 304428_S1_L002_R2_001.fastq.gz.U.qtrim ILLUMINACLIP:/gscratch/srlab/programs/trinity/trinity-plugins/Trimmomatic/adapters/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25 TrimmomaticPE: Started with arguments: -threads 28 -phred33 /gscratch/srlab/sr320/data/crab/304428_S1_L002_R1_001.fastq.gz /gscratch/srlab/sr320/data/crab/304428_S1_L002_R2_001.fastq.gz 304428_S1_L002_R1_001.fastq.gz.P.qtrim 304428_S1_L002_R1_001.fastq.gz.U.qtrim 304428_S1_L002_R2_001.fastq.gz.P.qtrim 304428_S1_L002_R2_001.fastq.gz.U.qtrim ILLUMINACLIP:/gscratch/srlab/programs/trinity/trinity-plugins/Trimmomatic/adapters/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25 Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT' ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences Input Read Pairs: 30285346 Both Surviving: 29481282 (97.35%) Forward Only Surviving: 801034 (2.64%) Reverse Only Surviving: 0 (0.00%) Dropped: 3030 (0.01%) TrimmomaticPE: Completed successfully Friday, October 26, 2018: 06:27:58 CMD: cp 304428_S1_L002_R1_001.fastq.gz.P.qtrim 304428_S1_L002_R1_001.fastq.gz.PwU.qtrim.fq Friday, October 26, 2018: 06:28:04 CMD: cp 304428_S1_L002_R2_001.fastq.gz.P.qtrim 304428_S1_L002_R2_001.fastq.gz.PwU.qtrim.fq Friday, October 26, 2018: 06:28:09 CMD: touch trimmomatic.ok Friday, October 26, 2018: 06:28:09 CMD: gzip 304428_S1_L002_R1_001.fastq.gz.P.qtrim 304428_S1_L002_R1_001.fastq.gz.U.qtrim 304428_S1_L002_R2_001.fastq.gz.P.qtrim 304428_S1_L002_R2_001.fastq.gz.U.qtrim & --------------------------------------------------------------- ------------ In silico Read Normalization --------------------- -- (Removing Excess Reads Beyond 50 Coverage -- --------------------------------------------------------------- # running normalization on reads: $VAR1 = [ [ '304428_S1_L001_R1_001.fastq.gz.PwU.qtrim.fq', '304428_S1_L002_R1_001.fastq.gz.PwU.qtrim.fq' ], [ '304428_S1_L001_R2_001.fastq.gz.PwU.qtrim.fq', '304428_S1_L002_R2_001.fastq.gz.PwU.qtrim.fq' ] ]; ####################################################################### Inchworm file: /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/inchworm.K25.L25.DS.fa detected. Skipping Inchworm Step, Using Previous Inchworm Assembly ####################################################################### -------------------------------------------------------- -------------------- Chrysalis ------------------------- -- (Contig Clustering & de Bruijn Graph Construction) -- -------------------------------------------------------- inchworm_target: /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/both.fa bowite_reads_fa: /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/both.fa chrysalis_reads_fa: /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/both.fa -- Skipping CMD: /gscratch/srlab/programs/trinity/util/support_scripts/filter_iworm_by_min_length_or_cov.pl /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/inchworm.K25.L25.DS.fa 100 10 > /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/inchworm.K25.L25.DS.fa.min100, checkpoint exists. -- Skipping CMD: bowtie2-build -o 3 /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/inchworm.K25.L25.DS.fa.min100 /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/inchworm.K25.L25.DS.fa.min100 1>/dev/null, checkpoint exists. -- Skipping CMD: bash -c " set -o pipefail;bowtie2 --local -k 2 --threads 28 -f --score-min G,46,0 -x /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/inchworm.K25.L25.DS.fa.min100 /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/both.fa | samtools view -@ 28 -F4 -Sb - | samtools sort -m 1917396114 -@ 28 -no - - > /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/iworm.bowtie.nameSorted.bam" , checkpoint exists. -- Skipping CMD: /gscratch/srlab/programs/trinity/util/support_scripts/scaffold_iworm_contigs.pl /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/iworm.bowtie.nameSorted.bam /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/inchworm.K25.L25.DS.fa > /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/iworm_scaffolds.txt, checkpoint exists. -- Skipping CMD: /gscratch/srlab/programs/trinity/Chrysalis/GraphFromFasta -i /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/inchworm.K25.L25.DS.fa -r /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/both.fa -min_contig_length 200 -min_glue 2 -glue_factor 0.05 -min_iso_ratio 0.05 -t 28 -k 24 -kk 48 -scaffolding /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/iworm_scaffolds.txt > /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/iworm_cluster_welds_graph.txt, checkpoint exists. -- Skipping CMD: /gscratch/srlab/programs/trinity/Chrysalis/BubbleUpClustering -i /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/inchworm.K25.L25.DS.fa -weld_graph /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/iworm_cluster_welds_graph.txt -min_contig_length 200 > /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/GraphFromIwormFasta.out, checkpoint exists. -- Skipping CMD: /gscratch/srlab/programs/trinity/Chrysalis/CreateIwormFastaBundle -i /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/GraphFromIwormFasta.out -o /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/bundled_iworm_contigs.fasta -min 200, checkpoint exists. -- Skipping CMD: /gscratch/srlab/programs/trinity/Chrysalis/ReadsToTranscripts -i /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/both.fa -f /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/bundled_iworm_contigs.fasta -o /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/readsToComponents.out -t 28 -max_mem_reads 50000000 , checkpoint exists. -- Skipping CMD: /usr/bin/sort --parallel=28 -T . -S 100G -k 1,1n /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/readsToComponents.out > /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/chrysalis/readsToComponents.out.sort, checkpoint exists. -------------------------------------------------------------------------------- ------------ Trinity Phase 2: Assembling Clusters of Reads --------------------- -------------------------------------------------------------------------------- Friday, October 26, 2018: 06:28:09 CMD: /gscratch/srlab/programs/trinity/trinity-plugins/parafly/bin/ParaFly -c recursive_trinity.cmds -CPU 28 -v warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c0.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c0.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c1.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c1.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c2.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c2.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c3.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c3.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c4.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c4.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c5.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c5.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c6.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c6.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c7.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c7.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c8.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c8.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c9.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c9.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c10.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c10.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c11.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c11.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c12.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c12.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c13.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c13.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. warning, command: /gscratch/srlab/programs/trinity/util/support_scripts/../../Trinity --single "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c14.trinity.reads.fa" --output "/gscratch/srlab/sr320/analyses/1026/trinity_out_dir/read_partitions/Fb_0/CBin_0/c14.trinity.reads.fa.out" --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup has successfully completed from a previous run. Skipping it here. Number of Commands: 0 All commands completed successfully. :-) ** Harvesting all assembled transcripts into a single multi-fasta file... Friday, October 26, 2018: 06:28:10 CMD: find read_partitions/ -name '*inity.fasta' | /gscratch/srlab/programs/trinity/util/support_scripts/partitioned_trinity_aggregator.pl TRINITY_DN > Trinity.fasta.tmp ################################################################### Butterfly assemblies are written to /gscratch/srlab/sr320/analyses/1026/trinity_out_dir/Trinity.fasta ###################################################################