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Teichlab / bracer

BraCeR - reconstruction of B cell receptor sequences from single-cell RNAseq data
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Bracer test failing at Trinity step with empty read files #11

Closed transcriptomics closed 5 years ago

transcriptomics commented 6 years ago

$ bracer test -p 16 -c ./bracer.conf -o ./test

Finding recombinant-derived reads

Attempting new assembly for ['BCR_H', 'BCR_K', 'BCR_L']

Detected average R1 read length: 50.0 Short read length detected. BraCeR will run two rounds of alignment for heavy chain

BCR_H

56438 reads; of these: 56438 (100.00%) were paired; of these: 35350 (62.64%) aligned concordantly 0 times 21088 (37.36%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times

35350 pairs aligned concordantly 0 times; of these:
  73 (0.21%) aligned discordantly 1 time
----
35277 pairs aligned 0 times concordantly or discordantly; of these:
  70554 mates make up the pairs; of these:
    69268 (98.18%) aligned 0 times
    232 (0.33%) aligned exactly 1 time
    1054 (1.49%) aligned >1 times

38.63% overall alignment rate 56438 reads; of these: 56438 (100.00%) were paired; of these: 56438 (100.00%) aligned concordantly 0 times 0 (0.00%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times

56438 pairs aligned concordantly 0 times; of these:
  0 (0.00%) aligned discordantly 1 time
----
56438 pairs aligned 0 times concordantly or discordantly; of these:
  112876 mates make up the pairs; of these:
    112876 (100.00%) aligned 0 times
    0 (0.00%) aligned exactly 1 time
    0 (0.00%) aligned >1 times

0.00% overall alignment rate

BCR_K

56438 reads; of these: 56438 (100.00%) were paired; of these: 48319 (85.61%) aligned concordantly 0 times 8119 (14.39%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times

48319 pairs aligned concordantly 0 times; of these:
  114 (0.24%) aligned discordantly 1 time
----
48205 pairs aligned 0 times concordantly or discordantly; of these:
  96410 mates make up the pairs; of these:
    95663 (99.23%) aligned 0 times
    195 (0.20%) aligned exactly 1 time
    552 (0.57%) aligned >1 times

15.25% overall alignment rate

BCR_L

56438 reads; of these: 56438 (100.00%) were paired; of these: 31766 (56.28%) aligned concordantly 0 times 24672 (43.72%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times

31766 pairs aligned concordantly 0 times; of these:
  270 (0.85%) aligned discordantly 1 time
----
31496 pairs aligned 0 times concordantly or discordantly; of these:
  62992 mates make up the pairs; of these:
    61549 (97.71%) aligned 0 times
    144 (0.23%) aligned exactly 1 time
    1299 (2.06%) aligned >1 times

45.47% overall alignment rate

Assembling Trinity Contigs

BCR_H

 ______  ____   ____  ____   ____  ______  __ __
|      ||    \ |    ||    \ |    ||      ||  |  |
|      ||  D  ) |  | |  _  | |  | |      ||  |  |
|_|  |_||    /  |  | |  |  | |  | |_|  |_||  ~  |
  |  |  |    \  |  | |  |  | |  |   |  |  |___, |
  |  |  |  .  \ |  | |  |  | |  |   |  |  |     |
  |__|  |__|\_||____||__|__||____|  |__|  |____/

Left read files: $VAR1 = [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_1.fastq' ]; Right read files: $VAR1 = [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_2.fastq' ]; Trinity version: __BLEEDING_EDGE__ ** NOTE: Latest version of Trinity is Trinity-v2.6.5, and can be obtained at: https://github.com/trinityrnaseq/trinityrnaseq/releases

Tuesday, February 13, 2018: 23:22:25 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /booleanfs/apps/trinityrnaseq-devel/util/support_scripts/ExitTester.jar 0 Tuesday, February 13, 2018: 23:22:25 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /booleanfs/apps/trinityrnaseq-devel/util/support_scripts/ExitTester.jar 1 Tuesday, February 13, 2018: 23:22:25 CMD: mkdir -p /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_H Tuesday, February 13, 2018: 23:22:25 CMD: mkdir -p /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_H/chrysalis

---------------------------------------------------------------------------------- -------------- Trinity Phase 1: Clustering of RNA-Seq Reads --------------------- ----------------------------------------------------------------------------------

--------------------------------------------------------------- ------------ In silico Read Normalization --------------------- -- (Removing Excess Reads Beyond 50 Coverage -- ---------------------------------------------------------------

# running normalization on reads: $VAR1 = [ [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_1.fastq' ], [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_2.fastq' ] ];

Tuesday, February 13, 2018: 23:22:25 CMD: /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl --seqType fq --JM 12G --max_cov 50 --min_cov 1 --CPU 16 --output /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_H/insilico_read_normalization --max_pct_stdev 10000 --left /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_1.fastq --right /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_2.fastq --pairs_together --PARALLEL_STATS
Converting input files. (both directions in parallel)CMD: seqtk-trinity seq -A /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_1.fastq >> left.fa CMD: seqtk-trinity seq -A /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_2.fastq >> right.fa CMD finished (0 seconds) CMD finished (0 seconds) Done converting input files.CMD: cat left.fa right.fa > both.fa CMD finished (0 seconds) ------------------------------------------- ----------- Jellyfish -------------------- -- (building a k-mer catalog from reads) -- -------------------------------------------

CMD: jellyfish count -t 16 -m 25 -s 100000000 --canonical both.fa CMD finished (2 seconds) CMD: jellyfish histo -t 16 -o jellyfish.K25.min2.kmers.fa.histo mer_counts.jf CMD finished (0 seconds) CMD: jellyfish dump -L 2 mer_counts.jf > jellyfish.K25.min2.kmers.fa CMD finished (0 seconds) CMD: touch jellyfish.K25.min2.kmers.fa.success CMD finished (0 seconds) CMD: /booleanfs/apps/trinityrnaseq-devel/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads left.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 8 --DS > left.fa.K25.stats CMD: /booleanfs/apps/trinityrnaseq-devel/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads right.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 8 --DS > right.fa.K25.stats -reading Kmer occurrences...-reading Kmer occurrences...

done parsing 0 Kmers, 0 added, taking 0 seconds. STATS_GENERATION_TIME: 0 seconds.

done parsing 0 Kmers, 0 added, taking 0 seconds. STATS_GENERATION_TIME: 0 seconds. CMD finished (0 seconds) CMD finished (0 seconds) -sorting each stats file by read name. CMD: /booleanfs/sysapps/coreutilsv8.29/bin/sort --parallel=16 -k5,5 -T . -S 6G left.fa.K25.stats > left.fa.K25.stats.sort CMD: /booleanfs/sysapps/coreutilsv8.29/bin/sort --parallel=16 -k5,5 -T . -S 6G right.fa.K25.stats > right.fa.K25.stats.sort CMD finished (0 seconds) CMD finished (0 seconds) CMD: /booleanfs/apps/trinityrnaseq-devel/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 (0 seconds) Error, pairs.K25.stats is empty. Be sure to check your fastq reads and ensure that the read names are identical except for the /1 or /2 designation. at /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl line 921. Error, cmd: /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl --seqType fq --JM 12G --max_cov 50 --min_cov 1 --CPU 16 --output /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_H/insilico_read_normalization --max_pct_stdev 10000 --left /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_1.fastq --right /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_H_2.fastq --pairs_together --PARALLEL_STATS died with ret 512 at /booleanfs/apps/trinityrnaseq-devel/Trinity line 2578. main::process_cmd("/booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normal"...) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 3124 main::normalize("/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinit"..., 50, ARRAY(0xb41d60), ARRAY(0xb41d48)) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 3071 main::run_normalization(50, ARRAY(0xb41d60), ARRAY(0xb41d48)) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 1291 Trinity failed for locus

BCR_K

 ______  ____   ____  ____   ____  ______  __ __
|      ||    \ |    ||    \ |    ||      ||  |  |
|      ||  D  ) |  | |  _  | |  | |      ||  |  |
|_|  |_||    /  |  | |  |  | |  | |_|  |_||  ~  |
  |  |  |    \  |  | |  |  | |  |   |  |  |___, |
  |  |  |  .  \ |  | |  |  | |  |   |  |  |     |
  |__|  |__|\_||____||__|__||____|  |__|  |____/

Left read files: $VAR1 = [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_1.fastq' ]; Right read files: $VAR1 = [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_2.fastq' ]; Trinity version: __BLEEDING_EDGE__ ** NOTE: Latest version of Trinity is Trinity-v2.6.5, and can be obtained at: https://github.com/trinityrnaseq/trinityrnaseq/releases

Tuesday, February 13, 2018: 23:22:28 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /booleanfs/apps/trinityrnaseq-devel/util/support_scripts/ExitTester.jar 0 Tuesday, February 13, 2018: 23:22:28 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /booleanfs/apps/trinityrnaseq-devel/util/support_scripts/ExitTester.jar 1 Tuesday, February 13, 2018: 23:22:28 CMD: mkdir -p /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_K Tuesday, February 13, 2018: 23:22:28 CMD: mkdir -p /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_K/chrysalis

---------------------------------------------------------------------------------- -------------- Trinity Phase 1: Clustering of RNA-Seq Reads --------------------- ----------------------------------------------------------------------------------

--------------------------------------------------------------- ------------ In silico Read Normalization --------------------- -- (Removing Excess Reads Beyond 50 Coverage -- ---------------------------------------------------------------

# running normalization on reads: $VAR1 = [ [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_1.fastq' ], [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_2.fastq' ] ];

Tuesday, February 13, 2018: 23:22:28 CMD: /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl --seqType fq --JM 12G --max_cov 50 --min_cov 1 --CPU 16 --output /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_K/insilico_read_normalization --max_pct_stdev 10000 --left /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_1.fastq --right /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_2.fastq --pairs_together --PARALLEL_STATS
Converting input files. (both directions in parallel)CMD: seqtk-trinity seq -A /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_1.fastq >> left.fa CMD: seqtk-trinity seq -A /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_2.fastq >> right.fa CMD finished (0 seconds) CMD finished (0 seconds) CMD: touch left.fa.ok CMD finished (0 seconds) Done converting input files.CMD: cat left.fa right.fa > both.fa CMD finished (0 seconds) CMD: touch both.fa.ok CMD finished (0 seconds) ------------------------------------------- ----------- Jellyfish -------------------- -- (building a k-mer catalog from reads) -- -------------------------------------------

CMD: jellyfish count -t 16 -m 25 -s 100000000 --canonical both.fa CMD finished (2 seconds) CMD: jellyfish histo -t 16 -o jellyfish.K25.min2.kmers.fa.histo mer_counts.jf CMD finished (0 seconds) CMD: jellyfish dump -L 2 mer_counts.jf > jellyfish.K25.min2.kmers.fa CMD finished (0 seconds) CMD: touch jellyfish.K25.min2.kmers.fa.success CMD finished (0 seconds) CMD: /booleanfs/apps/trinityrnaseq-devel/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads left.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 8 --DS > left.fa.K25.stats CMD: /booleanfs/apps/trinityrnaseq-devel/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads right.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 8 --DS > right.fa.K25.stats -reading Kmer occurrences... -reading Kmer occurrences...

done parsing 4712 Kmers, 4712 added, taking 0 seconds.

done parsing 4712 Kmers, 4712 added, taking 0 seconds. STATS_GENERATION_TIME: 0 seconds. CMD finished (0 seconds) STATS_GENERATION_TIME: 0 seconds. CMD finished (0 seconds) CMD: touch left.fa.K25.stats.ok CMD finished (0 seconds) -sorting each stats file by read name. CMD: /booleanfs/sysapps/coreutilsv8.29/bin/sort --parallel=16 -k5,5 -T . -S 6G left.fa.K25.stats > left.fa.K25.stats.sort CMD: /booleanfs/sysapps/coreutilsv8.29/bin/sort --parallel=16 -k5,5 -T . -S 6G right.fa.K25.stats > right.fa.K25.stats.sort CMD finished (0 seconds) CMD finished (0 seconds) CMD: touch left.fa.K25.stats.sort.ok CMD finished (0 seconds) CMD: /booleanfs/apps/trinityrnaseq-devel/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 (0 seconds) Error, pairs.K25.stats is empty. Be sure to check your fastq reads and ensure that the read names are identical except for the /1 or /2 designation. at /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl line 921. Error, cmd: /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl --seqType fq --JM 12G --max_cov 50 --min_cov 1 --CPU 16 --output /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_K/insilico_read_normalization --max_pct_stdev 10000 --left /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_1.fastq --right /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_K_2.fastq --pairs_together --PARALLEL_STATS died with ret 512 at /booleanfs/apps/trinityrnaseq-devel/Trinity line 2578. main::process_cmd("/booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normal"...) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 3124 main::normalize("/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinit"..., 50, ARRAY(0x1555d30), ARRAY(0x1555d60)) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 3071 main::run_normalization(50, ARRAY(0x1555d30), ARRAY(0x1555d60)) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 1291 Trinity failed for locus

BCR_L

 ______  ____   ____  ____   ____  ______  __ __
|      ||    \ |    ||    \ |    ||      ||  |  |
|      ||  D  ) |  | |  _  | |  | |      ||  |  |
|_|  |_||    /  |  | |  |  | |  | |_|  |_||  ~  |
  |  |  |    \  |  | |  |  | |  |   |  |  |___, |
  |  |  |  .  \ |  | |  |  | |  |   |  |  |     |
  |__|  |__|\_||____||__|__||____|  |__|  |____/

Left read files: $VAR1 = [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_1.fastq' ]; Right read files: $VAR1 = [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_2.fastq' ]; Trinity version: __BLEEDING_EDGE__ ** NOTE: Latest version of Trinity is Trinity-v2.6.5, and can be obtained at: https://github.com/trinityrnaseq/trinityrnaseq/releases

Tuesday, February 13, 2018: 23:22:31 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /booleanfs/apps/trinityrnaseq-devel/util/support_scripts/ExitTester.jar 0 Tuesday, February 13, 2018: 23:22:31 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /booleanfs/apps/trinityrnaseq-devel/util/support_scripts/ExitTester.jar 1 Tuesday, February 13, 2018: 23:22:31 CMD: mkdir -p /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L Tuesday, February 13, 2018: 23:22:31 CMD: mkdir -p /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L/chrysalis

---------------------------------------------------------------------------------- -------------- Trinity Phase 1: Clustering of RNA-Seq Reads --------------------- ----------------------------------------------------------------------------------

--------------------------------------------------------------- ------------ In silico Read Normalization --------------------- -- (Removing Excess Reads Beyond 50 Coverage -- ---------------------------------------------------------------

# running normalization on reads: $VAR1 = [ [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_1.fastq' ], [ '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_2.fastq' ] ];

Tuesday, February 13, 2018: 23:22:31 CMD: /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl --seqType fq --JM 12G --max_cov 50 --min_cov 1 --CPU 16 --output /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L/insilico_read_normalization --max_pct_stdev 10000 --left /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_1.fastq --right /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_2.fastq --pairs_together --PARALLEL_STATS
Converting input files. (both directions in parallel)CMD: seqtk-trinity seq -A /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_1.fastq >> left.fa CMD: seqtk-trinity seq -A /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_2.fastq >> right.fa CMD finished (0 seconds) CMD finished (0 seconds) CMD: touch left.fa.ok CMD finished (0 seconds) Done converting input files.CMD: cat left.fa right.fa > both.fa CMD finished (0 seconds) CMD: touch both.fa.ok CMD finished (0 seconds) ------------------------------------------- ----------- Jellyfish -------------------- -- (building a k-mer catalog from reads) -- -------------------------------------------

CMD: jellyfish count -t 16 -m 25 -s 100000000 --canonical both.fa CMD finished (2 seconds) CMD: jellyfish histo -t 16 -o jellyfish.K25.min2.kmers.fa.histo mer_counts.jf CMD finished (0 seconds) CMD: jellyfish dump -L 2 mer_counts.jf > jellyfish.K25.min2.kmers.fa CMD finished (0 seconds) CMD: touch jellyfish.K25.min2.kmers.fa.success CMD finished (0 seconds) CMD: /booleanfs/apps/trinityrnaseq-devel/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads left.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 8 --DS > left.fa.K25.stats CMD: /booleanfs/apps/trinityrnaseq-devel/util/..//Inchworm/bin/fastaToKmerCoverageStats --reads right.fa --kmers jellyfish.K25.min2.kmers.fa --kmer_size 25 --num_threads 8 --DS > right.fa.K25.stats -reading Kmer occurrences... -reading Kmer occurrences...

done parsing 13066 Kmers, 13066 added, taking 0 seconds.

done parsing 13066 Kmers, 13066 added, taking 0 seconds. STATS_GENERATION_TIME: 0 seconds. CMD finished (0 seconds) STATS_GENERATION_TIME: 0 seconds. CMD finished (0 seconds) CMD: touch left.fa.K25.stats.ok CMD finished (0 seconds) -sorting each stats file by read name. CMD: /booleanfs/sysapps/coreutilsv8.29/bin/sort --parallel=16 -k5,5 -T . -S 6G left.fa.K25.stats > left.fa.K25.stats.sort CMD: /booleanfs/sysapps/coreutilsv8.29/bin/sort --parallel=16 -k5,5 -T . -S 6G right.fa.K25.stats > right.fa.K25.stats.sort CMD finished (0 seconds) CMD finished (0 seconds) CMD: touch left.fa.K25.stats.sort.ok CMD finished (0 seconds) CMD: /booleanfs/apps/trinityrnaseq-devel/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 (0 seconds) Error, pairs.K25.stats is empty. Be sure to check your fastq reads and ensure that the read names are identical except for the /1 or /2 designation. at /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl line 921. Error, cmd: /booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normalization.pl --seqType fq --JM 12G --max_cov 50 --min_cov 1 --CPU 16 --output /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L/insilico_read_normalization --max_pct_stdev 10000 --left /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_1.fastq --right /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/aligned_reads/cell1_BCR_L_2.fastq --pairs_together --PARALLEL_STATS died with ret 512 at /booleanfs/apps/trinityrnaseq-devel/Trinity line 2578. main::process_cmd("/booleanfs/apps/trinityrnaseq-devel/util/insilico_read_normal"...) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 3124 main::normalize("/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinit"..., 50, ARRAY(0xf14df0), ARRAY(0xf14dd8)) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 3071 main::run_normalization(50, ARRAY(0xf14df0), ARRAY(0xf14dd8)) called at /booleanfs/apps/trinityrnaseq-devel/Trinity line 1291 Trinity failed for locus No successful Trinity assemblies

No recombinants found

idalind commented 6 years ago

Hi, I see that you are using the BLEEDING_EDGE version of Trinity. I'm not sure exactly which version this is and how old it is compared to Trinity-v2.4.0. BraCeR expects a Trinity version in the format of Trinity-v2.x.x in order to detect correct version. Could you please try to re-run the test using Trinity-v2.4.0? I will test BraCeR with later versions of Trinity and try to make it compatible with these versions as well.

transcriptomics commented 6 years ago

Thank you so much for your prompt reply. I am using the latest build of Trinity (2.6.5), which runs fine on other datasets. Older Trinity fails on my run environment.

The reason it says bleeding edge is because I had pulled and built Trinity from the devel branch at GitHub hours before the latest production release.

Btw Tracer runs fine when I issue “tracer test”.

From: idalind Sent: Wednesday, February 14, 2018 1:01 AM To: Teichlab/bracer Cc: transcriptomics; Author Subject: Re: [Teichlab/bracer] Bracer test failing at Trinity step with emptyread files (#11)

Hi, I see that you are using the BLEEDING_EDGE version of Trinity. I'm not sure exactly which version this is and how old it is compared to Trinity-v2.4.0. BraCeR expects a Trinity version in the format of Trinity-v2.x.x in order to detect correct version. Could you please try to re-run the test using Trinity-v2.4.0? I will test BraCeR with later versions of Trinity and try to make it compatible with these versions as well. — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub, or mute the thread.

idalind commented 6 years ago

Hi, I suspect this is an issue due to the in silico normalization not being turned off since BraCeR couldn't detect the Trinity version due to it's unexpected name. Could you please pull the newest commit for BraCeR (just updated) and add trinity_version = BLEEDING_EDGE under [trinity_options] in your config file? This should now be detected by BraCeR and in silico normalization will be turned off. Hopefully this will solve the issue with Trinity (although I cannot guarantee it since I haven't tested BraCeR for the newest versions). Please do let me know if it still doesn't work.

Best, Ida

transcriptomics commented 6 years ago

I simply pulled the official release of Trinity 2.6.5 and after that no error while running Trinity-- bracer detected the 2.x.x format of Trinity version.

However, the latest version of kallisto no longer has "--make-unique" option by default while making kallisto index. So I got a new error now while kallisto index was being made.

$ bracer test -p 12 -c ./bracer.conf -o ./test

Finding recombinant-derived reads

. . . . . . Thursday, February 15, 2018: 23:18:21 CMD: touch recursive_trinity.cmds.ok Thursday, February 15, 2018: 23:18:21 CMD: touch recursive_trinity.cmds.ok

-------------------------------------------------------------------------------- ------------ Trinity Phase 2: Assembling Clusters of Reads --------------------- --------------------------------------------------------------------------------

Thursday, February 15, 2018: 23:18:21 CMD: /booleanfs/apps/trinityrnaseqv2.6.5/trinity-plugins/BIN/ParaFly -c recursive_trinity.cmds -CPU 12 -v -shuffle Number of Commands: 1 succeeded(1) 100% completed.

All commands completed successfully. :-)

** Harvesting all assembled transcripts into a single multi-fasta file...

Thursday, February 15, 2018: 23:18:33 CMD: find /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L/read_partitions/ -name '*inity.fasta' | /booleanfs/apps/trinityrnaseqv2.6.5/util/support_scripts/partitioned_trinity_aggregator.pl TRINITY_DN > /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L/Trinity.fasta.tmp Thursday, February 15, 2018: 23:18:33 CMD: mv /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L/Trinity.fasta.tmp /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L.Trinity.fasta

################################################################### Butterfly assemblies are written to /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L.Trinity.fasta ###################################################################

Thursday, February 15, 2018: 23:18:33 CMD: /booleanfs/apps/trinityrnaseqv2.6.5/util/support_scripts/get_Trinity_gene_to_trans_map.pl /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L.Trinity.fasta > /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/Trinity_output/Trinity_cell1_BCR_L.Trinity.fasta.gene_trans_map

Running BLAST

Performing Blast on ['BCR_H', 'BCR_K', 'BCR_L']

BCR_H

BCR_K

BCR_L

Running IgBLAST

Ig_seqtype: Ig Performing IgBlast on ['BCR_H', 'BCR_K', 'BCR_L']

BCR_H

BCR_K

BCR_L

     START> MakeDB
   ALIGNER> IgBlast

ALIGNER_OUTPUT> cell1_BCR_K.fmt7 SEQ_FILE> cell1_BCR_K.Trinity.fasta NO_PARSE> False PARTIAL> False SCORES> True REGIONS> True

PROGRESS> [23:18:46] Done 0.0 min

PROGRESS> 23:18:46 |####################| 100% (1) 0.0 min

OUTPUT> /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/IgBLAST_output/cell1_BCR_K_db-pass.tab PASS> 1 FAIL> 0 END> MakeDb

     START> MakeDB
   ALIGNER> IgBlast

ALIGNER_OUTPUT> cell1_BCR_L.fmt7 SEQ_FILE> cell1_BCR_L.Trinity.fasta NO_PARSE> False PARTIAL> False SCORES> True REGIONS> True

PROGRESS> [23:18:47] Done 0.0 min

PROGRESS> 23:18:47 |####################| 100% (1) 0.0 min

OUTPUT> /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/IgBLAST_output/cell1_BCR_L_db-pass.tab PASS> 1 FAIL> 0 END> MakeDb

Running Kallisto

Making Kallisto indices

[build] loading fasta file /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/expression_quantification/kallisto_index/cell1_transcriptome.fa [build] k-mer length: 31 Error: repeated name in FASTA file /booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/expression_quantification/kallisto_index/cell1_transcriptome.fa ENST00000399012

Run with --make-unique to replace repeated names with unique names Traceback (most recent call last): File "/booleanfs/sysapps/Pythonv3.6.4/bin/bracer", line 11, in load_entry_point('bracer==0.1', 'console_scripts', 'bracer')() File "/booleanfs/sysapps/Pythonv3.6.4/lib/python3.6/site-packages/bracer-0.1-py3.6.egg/bracerlib/launcher.py", line 43, in launch Task().run() File "/booleanfs/sysapps/Pythonv3.6.4/lib/python3.6/site-packages/bracer-0.1-py3.6.egg/bracerlib/tasks.py", line 1941, in run trimmed_fastq2=self.trimmed_fastq2).run() File "/booleanfs/sysapps/Pythonv3.6.4/lib/python3.6/site-packages/bracer-0.1-py3.6.egg/bracerlib/tasks.py", line 369, in run self.quantify(cell) File "/booleanfs/sysapps/Pythonv3.6.4/lib/python3.6/site-packages/bracer-0.1-py3.6.egg/bracerlib/tasks.py", line 605, in quantify self.trimmed_fastq2, self.keep_trimmed_reads) File "/booleanfs/sysapps/Pythonv3.6.4/lib/python3.6/site-packages/bracer-0.1-py3.6.egg/bracerlib/bracer_func.py", line 2110, in quantify_with_kallisto subprocess.check_call(index_command) File "/booleanfs/sysapps/Pythonv3.6.4/lib/python3.6/subprocess.py", line 291, in check_call raise CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command '['/booleanfs/apps/kallisto_v0.44.0/kallisto', 'index', '-i', '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/expression_quantification/kallisto_index/cell1_transcriptome.idx', '/booleanfs/apps/Python3_pkgs/bracer/test/results/cell1/expression_quantification/kallisto_index/cell1_transcriptome.fa']' returned non-zero exit status 1.

idalind commented 6 years ago

@transcriptomics could you please try to use an earlier version of Kallisto until I have fixed compatibility with the newest version? I am currently using Kallisto v.0.43.0, so this should work.

Best, Ida

transcriptomics commented 6 years ago

Oh, no worries at all. In the script "bracer_func.py" I changed the line#2109 to

index_command = [kallisto, 'index', '--make-unique', '-i', idx_file, output_transcriptome]

And everything works fine. I did not want to change kallisto version as other things in my pipeline might have issues. Thanks again for looking into it.