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TraCeR - reconstruction of T cell receptor sequences from single-cell RNAseq data
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Error when running the test #55

Closed anne-marie-lyne closed 7 years ago

anne-marie-lyne commented 7 years ago

Hi,

I'm trying to run Tracer on my work cluster. It seems to run fine up until the part where it is quantifying with kallisto (or perhaps just after). Below is the last part of the error message. Any thoughts on what the problem could be?

Thanks, Anne-Marie

. . .

Quantifying with Kallisto

[quant] fragment length distribution will be estimated from the data [index] k-mer length: 31 [index] number of targets: 109,377 [index] number of k-mers: 95,790,004 [index] number of equivalence classes: 392,009 [quant] running in paired-end mode [quant] will process pair 1: /bioinfo/users/alyne/tracer-master/test_data/cell1_1.fastq /bioinfo/users/alyne/tracer-master/test_data/cell1_2.fastq [quant] finding pseudoalignments for the reads ... done [quant] processed 1,135 reads, 1,042 reads pseudoaligned [quant] estimated average fragment length: 106.333 [ em] quantifying the abundances ... done [ em] the Expectation-Maximization algorithm ran for 52 rounds

Traceback (most recent call last): File "tracer", line 21, in launch() File "/bioinfo/users/alyne/tracer-master/tracerlib/launcher.py", line 43, in launch Task().run() File "/bioinfo/users/alyne/tracer-master/tracerlib/tasks.py", line 1198, in run loci=['A', 'B'], max_junc_len=50).run() File "/bioinfo/users/alyne/tracer-master/tracerlib/tasks.py", line 369, in run self.quantify(cell) File "/bioinfo/users/alyne/tracer-master/tracerlib/tasks.py", line 635, in quantify tpm = counts[receptor][locus][rec.contig_name] KeyError: 'c0_g1_i2'

mstubb commented 7 years ago

Hi Anne-Marie,

What version of Trinity are you using? Would you mind sending me your config file and a zip of the output data for Cell1 in the test output directory?

Thanks!

mstubb commented 7 years ago

One more question: when you downloaded the reference mouse transcriptome, did you leave it as a zipped file rather than a plain-text fasta? If so, please unzip it and try again. This was the cause of this error previously so it might be again.

anne-marie-lyne commented 7 years ago

Hi Mike,

Thanks for your response.

The mouse transcriptome is unzipped so I don’t think it’s that. I attach a zip of the cell1 directory and a copy of the config file. I have the paths to all the required software in my .profile. The Trinity version says release 2014-07-17.

Anne-Marie

On 19 Oct 2017, at 17:44, Mike Stubbington notifications@github.com wrote:

One more question: when you downloaded the reference mouse transcriptome, did you leave it as a zipped file rather than a plain-text fasta? If so, please unzip it and try again. This was the cause of this error previously so it might be again.

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/Teichlab/tracer/issues/55#issuecomment-337949631, or mute the thread https://github.com/notifications/unsubscribe-auth/AFP2Um0TT4Xse7bORtVYUwyKut6QEFEtks5st25QgaJpZM4P6vnp.

mstubb commented 7 years ago

Thanks Anne-Marie. I think that Github has not been able to send me the zip file. Please could you send it directly to ms31@sanger.ac.uk ?

However, I think that the problem might be that you're using a very old version of Trinity. Are you able to update to the most recent version? If not, can you use the new Docker container to run Tracer (https://github.com/teichlab/tracer#docker-image)?

Thanks,

Mike

mstubb commented 7 years ago

@anne-marie-lyne sent the zip directly. For completeness and future reference, I'm moving this back to Github Issues. The email message is below:

Hi Mike,

Sorry for the delay. I installed the most recent version of Trinity and also updated the version of bowtie2. This has changed the error message slightly but I still get one. I copy the whole output below incase it is helpful. There is also an error message in the middle just before running igblast.

I attach the zipped cell1 directory.

Thanks again for your help,
Anne-Marie

##Finding recombinant-derived reads##
Attempting new assembly for ['TCR_A', 'TCR_B']

##TCR_A##
1135 reads; of these:
  1135 (100.00%) were paired; of these:
    571 (50.31%) aligned concordantly 0 times
    564 (49.69%) aligned concordantly exactly 1 time
    0 (0.00%) aligned concordantly >1 times
    ----
    571 pairs aligned concordantly 0 times; of these:
      0 (0.00%) aligned discordantly 1 time
    ----
    571 pairs aligned 0 times concordantly or discordantly; of these:
      1142 mates make up the pairs; of these:
        1010 (88.44%) aligned 0 times
        0 (0.00%) aligned exactly 1 time
        132 (11.56%) aligned >1 times
55.51% overall alignment rate
##TCR_B##
1135 reads; of these:
  1135 (100.00%) were paired; of these:
    687 (60.53%) aligned concordantly 0 times
    448 (39.47%) aligned concordantly exactly 1 time
    0 (0.00%) aligned concordantly >1 times
    ----
    687 pairs aligned concordantly 0 times; of these:
      0 (0.00%) aligned discordantly 1 time
    ----
    687 pairs aligned 0 times concordantly or discordantly; of these:
      1374 mates make up the pairs; of these:
        1267 (92.21%) aligned 0 times
        7 (0.51%) aligned exactly 1 time
        100 (7.28%) aligned >1 times
44.19% overall alignment rate

##Assembling Trinity Contigs##
##TCR_A##

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

Left read files: $VAR1 = [
          '/data/users/alyne/tracer-master/test_data/results/cell1/aligned_reads/cell1_TCR_A_1.fastq'
        ];
Right read files: $VAR1 = [
          '/data/users/alyne/tracer-master/test_data/results/cell1/aligned_reads/cell1_TCR_A_2.fastq'
        ];
Trinity version: Trinity-v2.5.0
** NOTE: Latest version of Trinity is Trinity-v2.5.1, and can be obtained at:
https://github.com/trinityrnaseq/trinityrnaseq/releases

Friday, October 20, 2017: 15:20:58
CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/ExitTester.jar 0
Friday, October 20, 2017: 15:20:59
CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/ExitTester.jar 1
Friday, October 20, 2017: 15:20:59
CMD: mkdir -p /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A
Friday, October 20, 2017: 15:20:59
CMD: mkdir -p /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis

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

Converting input files. (in parallel)Friday, October 20, 2017: 15:20:59
CMD: cat /data/users/alyne/tracer-master/test_data/results/cell1/aligned_reads/cell1_TCR_A_1.fastq | seqtk-trinity seq -A - >> left.fa
Friday, October 20, 2017: 15:20:59
CMD: cat /data/users/alyne/tracer-master/test_data/results/cell1/aligned_reads/cell1_TCR_A_2.fastq | seqtk-trinity seq -A - >> right.fa
Friday, October 20, 2017: 15:20:59
CMD: touch left.fa.ok
Friday, October 20, 2017: 15:20:59
CMD: touch right.fa.ok
Friday, October 20, 2017: 15:20:59
CMD: touch left.fa.ok right.fa.ok
Friday, October 20, 2017: 15:20:59
CMD: cat left.fa right.fa > /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa
Friday, October 20, 2017: 15:20:59
CMD: touch /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa.ok
-------------------------------------------
----------- Jellyfish  --------------------
-- (building a k-mer catalog from reads) --
-------------------------------------------

* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/trinity-plugins/jellyfish/bin/jellyfish count -t 1 -m 25 -s 100000000  --canonical  /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/trinity-plugins/jellyfish/bin/jellyfish dump -L 1 mer_counts.jf > jellyfish.kmers.fa
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/trinity-plugins/jellyfish/bin/jellyfish histo -t 1 -o jellyfish.kmers.fa.histo mer_counts.jf
----------------------------------------------
--------------- Inchworm ---------------------
-- (Linear contig construction from k-mers) --
----------------------------------------------

* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Inchworm/bin//inchworm --kmers jellyfish.kmers.fa --run_inchworm -K 25 -L 25 --monitor 1   --DS  --num_threads 1  --PARALLEL_IWORM  > /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/inchworm.K25.L25.DS.fa.tmp
* Running CMD: mv /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/inchworm.K25.L25.DS.fa.tmp /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/inchworm.K25.L25.DS.fa
Friday, October 20, 2017: 15:21:02
CMD: touch /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/inchworm.K25.L25.DS.fa.finished
--------------------------------------------------------
-------------------- Chrysalis -------------------------
-- (Contig Clustering & de Bruijn Graph Construction) --
--------------------------------------------------------

inchworm_target: /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa
bowite_reads_fa: /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa
chrysalis_reads_fa: /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/filter_iworm_by_min_length_or_cov.pl /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/inchworm.K25.L25.DS.fa 100 10 > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/inchworm.K25.L25.DS.fa.min100
* Running CMD: bowtie2-build --threads 1 -o 3 /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/inchworm.K25.L25.DS.fa.min100 /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/inchworm.K25.L25.DS.fa.min100 1>/dev/null
* Running CMD: bash -c " set -o pipefail;bowtie2 --local -k 2 --threads 1 -f --score-min G,46,0 -x /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/inchworm.K25.L25.DS.fa.min100 /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa  | samtools view -@ 1 -F4 -Sb - | samtools sort -m 2684354560 -@ 1 -no - - > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/iworm.bowtie.nameSorted.bam" 
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/scaffold_iworm_contigs.pl /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/iworm.bowtie.nameSorted.bam /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/inchworm.K25.L25.DS.fa > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/iworm_scaffolds.txt
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Chrysalis/GraphFromFasta -i /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/inchworm.K25.L25.DS.fa -r /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa  -min_contig_length 200 -min_glue 2 -glue_factor 0.05 -min_iso_ratio 0.05 -t 1 -k 24 -kk 48  -scaffolding /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/iworm_scaffolds.txt  > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/iworm_cluster_welds_graph.txt
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Chrysalis/BubbleUpClustering -i /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/inchworm.K25.L25.DS.fa  -weld_graph /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/iworm_cluster_welds_graph.txt -min_contig_length 200  > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/GraphFromIwormFasta.out
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Chrysalis/CreateIwormFastaBundle -i /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/GraphFromIwormFasta.out -o /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/bundled_iworm_contigs.fasta -min 200
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Chrysalis/ReadsToTranscripts -i /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/both.fa -f /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/bundled_iworm_contigs.fasta -o /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/readsToComponents.out -t 1 -max_mem_reads 50000000 
* Running CMD: /usr/bin/sort -T . -S 5G -k 1,1n /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/readsToComponents.out > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/chrysalis/readsToComponents.out.sort
Friday, October 20, 2017: 15:21:06
CMD: mkdir -p /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/read_partitions/Fb_0/CBin_0
Friday, October 20, 2017: 15:21:06
CMD: touch /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/partitioned_reads.files.list.ok
Friday, October 20, 2017: 15:21:06
CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/partitioned_reads.files.list --CPU 1 --max_memory 1G  --run_as_paired  --seqType fa --trinity_complete --full_cleanup  > recursive_trinity.cmds
Friday, October 20, 2017: 15:21:06
CMD: touch recursive_trinity.cmds.ok
Friday, October 20, 2017: 15:21:06
CMD: touch recursive_trinity.cmds.ok

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

Friday, October 20, 2017: 15:21:06
CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/trinity-plugins/BIN/ParaFly -c recursive_trinity.cmds -CPU 1 -v -shuffle 
Number of Commands: 2
succeeded(2)   100% completed.    

All commands completed successfully. :-)

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

Friday, October 20, 2017: 15:21:18
CMD: find /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/read_partitions/ -name '*inity.fasta'  | /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/partitioned_trinity_aggregator.pl TRINITY_DN > /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/Trinity.fasta.tmp
Friday, October 20, 2017: 15:21:18
CMD: mv /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A/Trinity.fasta.tmp /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A.Trinity.fasta

###################################################################
Butterfly assemblies are written to /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A.Trinity.fasta
###################################################################

Friday, October 20, 2017: 15:21:18
CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/get_Trinity_gene_to_trans_map.pl /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A.Trinity.fasta > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_A.Trinity.fasta.gene_trans_map
shell-init: error retrieving current directory: getcwd: cannot access parent directories: No such file or directory
##TCR_B##

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

Left read files: $VAR1 = [
          '/data/users/alyne/tracer-master/test_data/results/cell1/aligned_reads/cell1_TCR_B_1.fastq'
        ];
Right read files: $VAR1 = [
          '/data/users/alyne/tracer-master/test_data/results/cell1/aligned_reads/cell1_TCR_B_2.fastq'
        ];
Trinity version: Trinity-v2.5.0
** NOTE: Latest version of Trinity is Trinity-v2.5.1, and can be obtained at:
https://github.com/trinityrnaseq/trinityrnaseq/releases

Friday, October 20, 2017: 15:21:20
CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/ExitTester.jar 0
Friday, October 20, 2017: 15:21:20
CMD: java -Xmx64m -XX:ParallelGCThreads=2  -jar /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/ExitTester.jar 1
Friday, October 20, 2017: 15:21:20
CMD: mkdir -p /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B
Friday, October 20, 2017: 15:21:20
CMD: mkdir -p /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis

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

Converting input files. (in parallel)Friday, October 20, 2017: 15:21:20
CMD: cat /data/users/alyne/tracer-master/test_data/results/cell1/aligned_reads/cell1_TCR_B_1.fastq | seqtk-trinity seq -A - >> left.fa
Friday, October 20, 2017: 15:21:20
CMD: touch left.fa.ok
Friday, October 20, 2017: 15:21:20
CMD: cat /data/users/alyne/tracer-master/test_data/results/cell1/aligned_reads/cell1_TCR_B_2.fastq | seqtk-trinity seq -A - >> right.fa
Friday, October 20, 2017: 15:21:20
CMD: touch right.fa.ok
Friday, October 20, 2017: 15:21:20
CMD: touch left.fa.ok right.fa.ok
Friday, October 20, 2017: 15:21:20
CMD: cat left.fa right.fa > /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa
Friday, October 20, 2017: 15:21:20
CMD: touch /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa.ok
-------------------------------------------
----------- Jellyfish  --------------------
-- (building a k-mer catalog from reads) --
-------------------------------------------

* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/trinity-plugins/jellyfish/bin/jellyfish count -t 1 -m 25 -s 100000000  --canonical  /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/trinity-plugins/jellyfish/bin/jellyfish dump -L 1 mer_counts.jf > jellyfish.kmers.fa
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/trinity-plugins/jellyfish/bin/jellyfish histo -t 1 -o jellyfish.kmers.fa.histo mer_counts.jf
----------------------------------------------
--------------- Inchworm ---------------------
-- (Linear contig construction from k-mers) --
----------------------------------------------

* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Inchworm/bin//inchworm --kmers jellyfish.kmers.fa --run_inchworm -K 25 -L 25 --monitor 1   --DS  --num_threads 1  --PARALLEL_IWORM  > /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/inchworm.K25.L25.DS.fa.tmp
* Running CMD: mv /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/inchworm.K25.L25.DS.fa.tmp /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/inchworm.K25.L25.DS.fa
Friday, October 20, 2017: 15:21:24
CMD: touch /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/inchworm.K25.L25.DS.fa.finished
--------------------------------------------------------
-------------------- Chrysalis -------------------------
-- (Contig Clustering & de Bruijn Graph Construction) --
--------------------------------------------------------

inchworm_target: /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa
bowite_reads_fa: /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa
chrysalis_reads_fa: /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/filter_iworm_by_min_length_or_cov.pl /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/inchworm.K25.L25.DS.fa 100 10 > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/inchworm.K25.L25.DS.fa.min100
* Running CMD: bowtie2-build --threads 1 -o 3 /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/inchworm.K25.L25.DS.fa.min100 /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/inchworm.K25.L25.DS.fa.min100 1>/dev/null
* Running CMD: bash -c " set -o pipefail;bowtie2 --local -k 2 --threads 1 -f --score-min G,46,0 -x /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/inchworm.K25.L25.DS.fa.min100 /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa  | samtools view -@ 1 -F4 -Sb - | samtools sort -m 2684354560 -@ 1 -no - - > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/iworm.bowtie.nameSorted.bam" 
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/scaffold_iworm_contigs.pl /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/iworm.bowtie.nameSorted.bam /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/inchworm.K25.L25.DS.fa > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/iworm_scaffolds.txt
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Chrysalis/GraphFromFasta -i /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/inchworm.K25.L25.DS.fa -r /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa  -min_contig_length 200 -min_glue 2 -glue_factor 0.05 -min_iso_ratio 0.05 -t 1 -k 24 -kk 48  -scaffolding /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/iworm_scaffolds.txt  > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/iworm_cluster_welds_graph.txt
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Chrysalis/BubbleUpClustering -i /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/inchworm.K25.L25.DS.fa  -weld_graph /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/iworm_cluster_welds_graph.txt -min_contig_length 200  > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/GraphFromIwormFasta.out
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Chrysalis/CreateIwormFastaBundle -i /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/GraphFromIwormFasta.out -o /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/bundled_iworm_contigs.fasta -min 200
* Running CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/Chrysalis/ReadsToTranscripts -i /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/both.fa -f /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/bundled_iworm_contigs.fasta -o /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/readsToComponents.out -t 1 -max_mem_reads 50000000 
* Running CMD: /usr/bin/sort -T . -S 5G -k 1,1n /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/readsToComponents.out > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/chrysalis/readsToComponents.out.sort
Friday, October 20, 2017: 15:21:27
CMD: mkdir -p /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/read_partitions/Fb_0/CBin_0
Friday, October 20, 2017: 15:21:27
CMD: touch /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/partitioned_reads.files.list.ok
Friday, October 20, 2017: 15:21:27
CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/partitioned_reads.files.list --CPU 1 --max_memory 1G  --run_as_paired  --seqType fa --trinity_complete --full_cleanup  > recursive_trinity.cmds
Friday, October 20, 2017: 15:21:27
CMD: touch recursive_trinity.cmds.ok
Friday, October 20, 2017: 15:21:27
CMD: touch recursive_trinity.cmds.ok

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

Friday, October 20, 2017: 15:21:27
CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/trinity-plugins/BIN/ParaFly -c recursive_trinity.cmds -CPU 1 -v -shuffle 
Number of Commands: 1
succeeded(1)   100% completed.    

All commands completed successfully. :-)

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

Friday, October 20, 2017: 15:21:33
CMD: find /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/read_partitions/ -name '*inity.fasta'  | /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/partitioned_trinity_aggregator.pl TRINITY_DN > /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/Trinity.fasta.tmp
Friday, October 20, 2017: 15:21:33
CMD: mv /bioinfo/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B/Trinity.fasta.tmp /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B.Trinity.fasta

###################################################################
Butterfly assemblies are written to /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B.Trinity.fasta
###################################################################

Friday, October 20, 2017: 15:21:33
CMD: /bioinfo/users/alyne/trinityrnaseq-Trinity-v2.5.0/util/support_scripts/get_Trinity_gene_to_trans_map.pl /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B.Trinity.fasta > /data/users/alyne/tracer-master/test_data/results/cell1/Trinity_output/Trinity_cell1_TCR_B.Trinity.fasta.gene_trans_map
shell-init: error retrieving current directory: getcwd: cannot access parent directories: No such file or directory

##Running IgBLAST##
##Running IgBLAST##
Performing IgBlast on ['TCR_A', 'TCR_B']
##TCR_A##
##TCR_B##

##Running Kallisto##
##Making Kallisto indices##

[build] loading fasta file /data/users/alyne/tracer-master/test_data/results/cell1/expression_quantification/kallisto_index/cell1_transcriptome.fa
[build] k-mer length: 31
[build] warning: clipped off poly-A tail (longer than 10)
        from 667 target sequences
[build] warning: replaced 41 non-ACGUT characters in the input sequence
        with pseudorandom nucleotides
[build] counting k-mers ... done.
[build] building target de Bruijn graph ...  done 
[build] creating equivalence classes ...  done
[build] target de Bruijn graph has 665193 contigs and contains 95790017 k-mers 

##Quantifying with Kallisto##

[quant] fragment length distribution will be estimated from the data
[index] k-mer length: 31
[index] number of targets: 109,377
[index] number of k-mers: 95,790,017
[index] number of equivalence classes: 392,009
[quant] running in paired-end mode
[quant] will process pair 1: /bioinfo/users/alyne/tracer-master/test_data/cell1_1.fastq
                             /bioinfo/users/alyne/tracer-master/test_data/cell1_2.fastq
[quant] finding pseudoalignments for the reads ... done
[quant] processed 1,135 reads, 1,042 reads pseudoaligned
[quant] estimated average fragment length: 106.333
[   em] quantifying the abundances ... done
[   em] the Expectation-Maximization algorithm ran for 52 rounds

Traceback (most recent call last):
  File "tracer", line 21, in <module>
    launch()
  File "/bioinfo/users/alyne/tracer-master/tracerlib/launcher.py", line 43, in launch
    Task().run()
  File "/bioinfo/users/alyne/tracer-master/tracerlib/tasks.py", line 1198, in run
    loci=['A', 'B'], max_junc_len=50).run()
  File "/bioinfo/users/alyne/tracer-master/tracerlib/tasks.py", line 369, in run
    self.quantify(cell)
  File "/bioinfo/users/alyne/tracer-master/tracerlib/tasks.py", line 635, in quantify
    tpm = counts[receptor][locus][rec.contig_name]
KeyError: 'TRINITY_DN0_c0_g1_i1'
mstubb commented 7 years ago

Thanks for this Anne-Marie. Please can you check if there is a line-break in your transcriptome reference after the final ERCC sequence.

From looking at your output, it seems that the first reconstructed TCRA sequence isn't being quantified by Kallisto which is possibly because it's not being properly appended to the reference transcriptome. This might be caused by a missing line break at the end of your reference file.

mstubb commented 7 years ago

PS - that error in the Trinity output seems to be caused by something new introduced in v2.5.0 but it doesn't have an effect on Trinity's use for TraCeR

anne-marie-lyne commented 7 years ago

Hi Mike,

Yes, it was the lack of line break at the end of the reference fasta that was the problem. The test is now working as expected. Thank you, I certainly would never have thought of that!

Kind regards, Anne-Marie

On 20 Oct 2017, at 17:52, Mike Stubbington notifications@github.com wrote:

PS - that error in the Trinity output seems to be caused by something new introduced in v2.5.0 but it doesn't have an effect on Trinity's use for TraCeR

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/Teichlab/tracer/issues/55#issuecomment-338246627, or mute the thread https://github.com/notifications/unsubscribe-auth/AFP2Uomfcg-sd1B28mRmTcamIbyZYkbhks5suMG4gaJpZM4P6vnp.

mstubb commented 7 years ago

Fantastic! Glad I could help.