Left read files: $VAR1 = [ '/gscratch/srlab/sr320/data/NR021_R1_comb.fastq' ]; Right read files: $VAR1 = [ '/gscratch/srlab/sr320/data/NR021_R2_comb.fastq' ]; Trinity version: Trinity-v2.4.0 -ERROR: couldn't run the network check to confirm latest Trinity software version. Friday, August 4, 2017: 10:34:33 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /gscratch/srlab/programs/trinity/util/support_scripts/ExitTester.jar 0 Friday, August 4, 2017: 10:34:33 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/NR021_R1_comb.fastq, /gscratch/srlab/sr320/data/NR021_R2_comb.fastq Friday, August 4, 2017: 10:34:33 CMD: java -jar /gscratch/srlab/programs/trinity/trinity-plugins/Trimmomatic/trimmomatic.jar PE -threads 50 -phred33 /gscratch/srlab/sr320/data/NR021_R1_comb.fastq /gscratch/srlab/sr320/data/NR021_R2_comb.fastq NR021_R1_comb.fastq.P.qtrim NR021_R1_comb.fastq.U.qtrim NR021_R2_comb.fastq.P.qtrim NR021_R2_comb.fastq.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 50 -phred33 /gscratch/srlab/sr320/data/NR021_R1_comb.fastq /gscratch/srlab/sr320/data/NR021_R2_comb.fastq NR021_R1_comb.fastq.P.qtrim NR021_R1_comb.fastq.U.qtrim NR021_R2_comb.fastq.P.qtrim NR021_R2_comb.fastq.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: 7613170 Both Surviving: 5969058 (78.40%) Forward Only Surviving: 1639586 (21.54%) Reverse Only Surviving: 0 (0.00%) Dropped: 4526 (0.06%) TrimmomaticPE: Completed successfully Friday, August 4, 2017: 10:34:44 CMD: cp NR021_R1_comb.fastq.P.qtrim NR021_R1_comb.fastq.PwU.qtrim.fq Friday, August 4, 2017: 10:34:45 CMD: cp NR021_R2_comb.fastq.P.qtrim NR021_R2_comb.fastq.PwU.qtrim.fq Friday, August 4, 2017: 10:34:47 CMD: touch trimmomatic.ok Friday, August 4, 2017: 10:34:47 CMD: gzip NR021_R1_comb.fastq.P.qtrim NR021_R1_comb.fastq.U.qtrim NR021_R2_comb.fastq.P.qtrim NR021_R2_comb.fastq.U.qtrim & --------------------------------------------------------------- ------------ In silico Read Normalization --------------------- -- (Removing Excess Reads Beyond 50 Coverage -- --------------------------------------------------------------- gzip: NR021_R1_comb.fastq.P.qtrim.gz already exists; not overwritten gzip: NR021_R1_comb.fastq.U.qtrim.gz already exists; not overwritten # running normalization on reads: $VAR1 = [ [ 'NR021_R1_comb.fastq.PwU.qtrim.fq' ], [ 'NR021_R2_comb.fastq.PwU.qtrim.fq' ] ]; Friday, August 4, 2017: 10:34:47 CMD: /gscratch/srlab/programs/trinity/util/insilico_read_normalization.pl --seqType fq --JM 500G --max_cov 50 --CPU 50 --output /gscratch/srlab/sr320/analyses/trinity_out_dir/insilico_read_normalization --max_pct_stdev 10000 --left NR021_R1_comb.fastq.PwU.qtrim.fq --right NR021_R2_comb.fastq.PwU.qtrim.fq --pairs_together --PARALLEL_STATS Converting input files. (both directions in parallel)left file exists, nothing to doright file exists, nothing to do------------------------------------------- ----------- Jellyfish -------------------- -- (building a k-mer catalog from reads) -- ------------------------------------------- Done converting input files.CMD: /gscratch/srlab/programs/trinity/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads left.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 50 --DS > left.fa.K25.stats CMD: /gscratch/srlab/programs/trinity/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads right.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 50 --DS > right.fa.K25.stats -reading Kmer occurrences...-reading Kmer occurrences... done parsing 215744308 Kmers, 215744308 added, taking 247 seconds. done parsing 215744308 Kmers, 215744308 added, taking 256 seconds. STATS_GENERATION_TIME: 139 seconds. STATS_GENERATION_TIME: 145 seconds. CMD finished (432 seconds) CMD finished (446 seconds) CMD: touch left.fa.K25.stats.ok CMD finished (0 seconds) CMD: touch right.fa.K25.stats.ok -sorting each stats file by read name. CMD finished (0 seconds) CMD: /bin/sort --parallel=50 -k5,5 -T . -S 250G left.fa.K25.stats > left.fa.K25.stats.sort CMD: /bin/sort --parallel=50 -k5,5 -T . -S 250G right.fa.K25.stats > right.fa.K25.stats.sort CMD finished (4 seconds) CMD finished (4 seconds) CMD: touch left.fa.K25.stats.sort.ok CMD finished (0 seconds) CMD: touch right.fa.K25.stats.sort.ok CMD finished (0 seconds) CMD: /gscratch/srlab/programs/trinity/util/..//util/support_scripts//nbkc_merge_left_right_stats.pl --left left.fa.K25.stats.sort --right right.fa.K25.stats.sort --sorted > pairs.K25.stats -opening left.fa.K25.stats.sort -opening right.fa.K25.stats.sort -done opening files. CMD finished (39 seconds) CMD: touch pairs.K25.stats.ok CMD finished (0 seconds) CMD: /gscratch/srlab/programs/trinity/util/..//util/support_scripts//nbkc_normalize.pl pairs.K25.stats 50 10000 > pairs.K25.stats.C50.pctSD10000.accs 4659890 / 7613163 = 61.21% reads selected during normalization. 0 / 7613163 = 0.00% reads discarded as likely aberrant based on coverage profiles. 0 / 7613163 = 0.00% reads missing kmer coverage (N chars included?). CMD finished (12 seconds) CMD: touch pairs.K25.stats.C50.pctSD10000.accs.ok CMD finished (0 seconds) Thread 8 terminated abnormally: Error, not all specified records have been retrieved (missing 907086) from /gscratch/srlab/sr320/analyses/trinity_out_dir/NR021_R2_comb.fastq.PwU.qtrim.fq, see file: /gscratch/srlab/sr320/analyses/trinity_out_dir/insilico_read_normalization/NR021_R2_comb.fastq.PwU.qtrim.fq.normalized_K25_C50_pctSD10000.fq.missing_accs for list of missing entries at /gscratch/srlab/programs/trinity/util/insilico_read_normalization.pl line 548. Thread 7 terminated abnormally: Error, not all specified records have been retrieved (missing 907086) from /gscratch/srlab/sr320/analyses/trinity_out_dir/NR021_R1_comb.fastq.PwU.qtrim.fq, see file: /gscratch/srlab/sr320/analyses/trinity_out_dir/insilico_read_normalization/NR021_R1_comb.fastq.PwU.qtrim.fq.normalized_K25_C50_pctSD10000.fq.missing_accs for list of missing entries at /gscratch/srlab/programs/trinity/util/insilico_read_normalization.pl line 548. Error encountered with thread. Error encountered with thread. Error, at least one thread died at /gscratch/srlab/programs/trinity/util/insilico_read_normalization.pl line 419. Error, cmd: /gscratch/srlab/programs/trinity/util/insilico_read_normalization.pl --seqType fq --JM 500G --max_cov 50 --CPU 50 --output /gscratch/srlab/sr320/analyses/trinity_out_dir/insilico_read_normalization --max_pct_stdev 10000 --left NR021_R1_comb.fastq.PwU.qtrim.fq --right NR021_R2_comb.fastq.PwU.qtrim.fq --pairs_together --PARALLEL_STATS died with ret 6400 at /gscratch/srlab/programs/trinity/Trinity line 2462.