KeyError message in assemble #90
Comments
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Hi Stijn,
Are you using all built-in sequence references? It looks like the TRAV29_DV5*03 allele isn’t in https://github.com/Teichlab/tracer/blob/master/resources/Hsap/raw_seqs/TCR_A_V.fa <https://github.com/Teichlab/tracer/blob/master/resources/Hsap/raw_seqs/TCR_A_V.fa> so Tracer can’t extract the sequence to make a full-length version based on the reference. However, if it’s not in the reference, I’m a bit confused about why it’s being provided as an annotation by IgBLAST. I'd have to dive in and remember exactly how the references were set up.
In the meantime, can you run that cell with the `-m assembly` flag (detailed here https://github.com/Teichlab/tracer#options <https://github.com/Teichlab/tracer#options>) which I think will stop it trying to find the sequence in the references.
Let me know if any of that works. I’ll try to find time to dig in more but might not be for a little while, sorry.
Cheers,
Mike
… On 13 May 2019, at 18:32, micans ***@***.***> wrote:
Hi, below is the KeyError error that I get with tracer assemble. It is only one out of 1536 samples, so otherwise the software is running very nicely. Below the error I have copied a listing of the output files that were generated; in particular the fastq files are non-empty (they are uncompressed into the files f1 and f2). I notice that the 'overall alignment rate' looks low (0.12%), but it's similar in other samples. Is this something I need to worry about? Let me know if there is a way to dig further into this,
Thanks,
Stijn
Error
924078 reads; of these:
924078 (100.00%) were paired; of these:
923196 (99.90%) aligned concordantly 0 times
882 (0.10%) aligned concordantly exactly 1 time
0 (0.00%) aligned concordantly >1 times
----
923196 pairs aligned concordantly 0 times; of these:
22 (0.00%) aligned discordantly 1 time
----
923174 pairs aligned 0 times concordantly or discordantly; of these:
1846348 mates make up the pairs; of these:
1845935 (99.98%) aligned 0 times
321 (0.02%) aligned exactly 1 time
92 (0.00%) aligned >1 times
0.12% overall alignment rate
924078 reads; of these:
924078 (100.00%) were paired; of these:
922260 (99.80%) aligned concordantly 0 times
1818 (0.20%) aligned concordantly exactly 1 time
0 (0.00%) aligned concordantly >1 times
----
922260 pairs aligned concordantly 0 times; of these:
6 (0.00%) aligned discordantly 1 time
----
922254 pairs aligned 0 times concordantly or discordantly; of these:
1844508 mates make up the pairs; of these:
1843450 (99.94%) aligned 0 times
863 (0.05%) aligned exactly 1 time
195 (0.01%) aligned >1 times
0.25% overall alignment rate
Traceback (most recent call last):
File "/usr/local/bin/tracer", line 11, in <module>
load_entry_point('tracer==0.5', 'console_scripts', 'tracer')()
File "/usr/local/lib/python3.5/dist-packages/tracer-0.5-py3.5.egg/tracerlib/launcher.py", line 43, in launch
Task().run()
File "/usr/local/lib/python3.5/dist-packages/tracer-0.5-py3.5.egg/tracerlib/tasks.py", line 372, in run
cell = self.ig_blast()
File "/usr/local/lib/python3.5/dist-packages/tracer-0.5-py3.5.egg/tracerlib/tasks.py", line 515, in ig_blast
self.max_junc_len)
File "/usr/local/lib/python3.5/dist-packages/tracer-0.5-py3.5.egg/tracerlib/io.py", line 113, in parse_IgBLAST
loci, max_junc_len)
File "/usr/local/lib/python3.5/dist-packages/tracer-0.5-py3.5.egg/tracerlib/tracer_func.py", line 213, in find_possible_alignments
query_name, species, loci_for_segments)
File "/usr/local/lib/python3.5/dist-packages/tracer-0.5-py3.5.egg/tracerlib/tracer_func.py", line 382, in get_fasta_line_for_contig_imgt
best_V_seq = IMGT_seqs[V_locus_key][segment]
KeyError: 'TRAV29_DV5*03'
Directory structure:
***@***.***:/workspace/svd/work2/7d/140d7c90d67b9c1900e3ee491987da# find -maxdepth 3 -exec ls -l {} \;
total 336591
lrwxrwxrwx 1 root root 55 May 13 12:50 Human_colon_16S7722453_1.fastq.gz -> /workspace/svd/fastqs/Human_colon_16S7722453_1.fastq.gz
lrwxrwxrwx 1 root root 55 May 13 12:50 Human_colon_16S7722453_2.fastq.gz -> /workspace/svd/fastqs/Human_colon_16S7722453_2.fastq.gz
-rw-r--r-- 1 root root 172334567 May 13 12:50 f1
-rw-r--r-- 1 root root 172334567 May 13 12:50 f2
drwxr-xr-x 3 root root 48 May 13 12:50 out_asm
-rw-r--r-- 1 root root 235 May 13 12:50 ./.command.sh
-rw-r--r-- 1 root root 3484 May 13 12:50 ./.command.stub
-rw-r--r-- 1 root root 2274 May 13 12:50 ./.command.run
-rw-r--r-- 1 root root 717 May 13 12:50 ./.command.yaml
-rw-r--r-- 1 root root 0 May 13 12:50 ./.command.begin
lrwxrwxrwx 1 root root 55 May 13 12:50 ./Human_colon_16S7722453_1.fastq.gz -> /workspace/svd/fastqs/Human_colon_16S7722453_1.fastq.gz
lrwxrwxrwx 1 root root 55 May 13 12:50 ./Human_colon_16S7722453_2.fastq.gz -> /workspace/svd/fastqs/Human_colon_16S7722453_2.fastq.gz
-rw-r--r-- 1 root root 24373 May 13 12:50 ./.command.out
-rw-r--r-- 1 root root 2283 May 13 12:50 ./.command.err
-rw-r--r-- 1 root root 194 May 13 12:51 ./.command.trace
-rw-r--r-- 1 root root 172334567 May 13 12:50 ./f1
-rw-r--r-- 1 root root 172334567 May 13 12:50 ./f2
total 0
drwxr-xr-x 8 root root 184 May 13 12:50 out-Human_colon_16S7722453
total 4
drwxr-xr-x 2 root root 240 May 13 14:44 IgBLAST_output
drwxr-xr-x 2 root root 218 May 13 12:50 Trinity_output
drwxr-xr-x 2 root root 4096 May 13 12:50 aligned_reads
drwxr-xr-x 2 root root 10 May 13 12:50 expression_quantification
drwxr-xr-x 2 root root 10 May 13 12:50 filtered_TCR_seqs
drwxr-xr-x 2 root root 10 May 13 12:50 unfiltered_TCR_seqs
total 3499
-rw-r--r-- 1 root root 951335 May 13 12:50 out-Human_colon_16S7722453_TCR_A.sam
-rw-r--r-- 1 root root 177160 May 13 12:50 out-Human_colon_16S7722453_TCR_A_1.fastq
-rw-r--r-- 1 root root 177160 May 13 12:50 out-Human_colon_16S7722453_TCR_A_2.fastq
-rw-r--r-- 1 root root 1558060 May 13 12:50 out-Human_colon_16S7722453_TCR_B.sam
-rw-r--r-- 1 root root 358400 May 13 12:50 out-Human_colon_16S7722453_TCR_B_1.fastq
-rw-r--r-- 1 root root 358400 May 13 12:50 out-Human_colon_16S7722453_TCR_B_2.fastq
total 10
-rw-r--r-- 1 root root 5268 May 13 12:50 out-Human_colon_16S7722453_TCR_A.Trinity.fasta
-rw-r--r-- 1 root root 3261 May 13 12:50 out-Human_colon_16S7722453_TCR_B.Trinity.fasta
-rw-r--r-- 1 root root 51 May 13 12:50 successful_trinity_assemblies.txt
-rw-r--r-- 1 root root 0 May 13 12:50 unsuccessful_trinity_assemblies.txt
total 90
-rw-r--r-- 1 root root 15495 May 13 12:50 out-Human_colon_16S7722453_TCR_A.IgBLASTOut
-rw-r--r-- 1 root root 41591 May 13 12:50 out-Human_colon_16S7722453_TCR_A_fmt3.IgBLASTOut
-rw-r--r-- 1 root root 9746 May 13 12:50 out-Human_colon_16S7722453_TCR_B.IgBLASTOut
-rw-r--r-- 1 root root 23827 May 13 12:50 out-Human_colon_16S7722453_TCR_B_fmt3.IgBLASTOut
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Hi Mike, |
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Yes, I think that alignment rate looks about right. If you’re managing to assemble TCR sequences then it’s sufficient :)
Cheers,
Mike
… On 13 May 2019, at 18:55, micans ***@***.***> wrote:
Hi Mike,
thanks for the quick response and the suggestion to use -m assembly. I realised I missed out some bits; I'm using essentially the docker container teichlab/tracer with procps added. The command I run is this: tracer assemble -p 4 -s Hsap f1 f2 out-Human_colon_16S7722453 out_asm. It all works for the other 1535 samples (I just run the software by the way, I'm not the scientist). That said, I am a bit curious about the alignment rate: A random sample among those 1536 samples is below; is this roughly within the expected/acceptable range? If that's the case we'll be very happy!
Many thanks,
Stijn
./83/9ea943ed0fe638dcc2eddedd6b98b0/.command.err:0.20% overall alignment rate
./83/a011e1467d6d7613817e33c04df70a/.command.err:0.09% overall alignment rate
./83/a011e1467d6d7613817e33c04df70a/.command.err:0.15% overall alignment rate
./83/5b84fe14081acb64b8a7d750774663/.command.err:0.08% overall alignment rate
./83/5b84fe14081acb64b8a7d750774663/.command.err:0.09% overall alignment rate
./83/09e9fbfd1e15f7340b5f9f395cb5a2/.command.err:0.04% overall alignment rate
./83/09e9fbfd1e15f7340b5f9f395cb5a2/.command.err:0.16% overall alignment rate
./83/8f2f56732833a2e03d79ab7c140f7a/.command.err:0.05% overall alignment rate
./83/8f2f56732833a2e03d79ab7c140f7a/.command.err:0.13% overall alignment rate
./e9/8bfe761f4ab3e5a84306578261a2be/.command.err:0.09% overall alignment rate
./e9/8bfe761f4ab3e5a84306578261a2be/.command.err:0.17% overall alignment rate
./e9/913649ac6df02441b755a6d50026a2/.command.err:0.00% overall alignment rate
./e9/913649ac6df02441b755a6d50026a2/.command.err:0.01% overall alignment rate
./e9/91b84f6e97c558967ce95a176c06ba/.command.err:0.06% overall alignment rate
./e9/91b84f6e97c558967ce95a176c06ba/.command.err:0.17% overall alignment rate
./e9/65cfc6a310fc111fe4c7414669c04a/.command.err:0.08% overall alignment rate
./e9/65cfc6a310fc111fe4c7414669c04a/.command.err:0.05% overall alignment rate
./e9/61b455fe01d14a6a83952f5921ef24/.command.err:0.02% overall alignment rate
./e9/61b455fe01d14a6a83952f5921ef24/.command.err:0.14% overall alignment rate
./e9/d4ecfeaa2fc8bdc8a7baaff1203216/.command.err:0.03% overall alignment rate
./e9/d4ecfeaa2fc8bdc8a7baaff1203216/.command.err:0.09% overall alignment rate
./e9/de46e46aafd7bd876557461950c2fb/.command.err:0.03% overall alignment rate
./e9/de46e46aafd7bd876557461950c2fb/.command.err:0.26% overall alignment rate
./e9/e538aa7cb1f52022f17063961115b8/.command.err:0.07% overall alignment rate
./e9/e538aa7cb1f52022f17063961115b8/.command.err:0.21% overall alignment rate
./e9/1dca45b0f774af51837183795ef1b2/.command.err:0.06% overall alignment rate
./e9/1dca45b0f774af51837183795ef1b2/.command.err:0.07% overall alignment rate
./5d/92d6848531de1314472cc93fd925e6/.command.err:0.01% overall alignment rate
./5d/92d6848531de1314472cc93fd925e6/.command.err:0.01% overall alignment rate
./5d/d89652caa95d22bcab04a3967c4fe4/.command.err:0.12% overall alignment rate
./5d/d89652caa95d22bcab04a3967c4fe4/.command.err:0.11% overall alignment rate
./5d/4bd31c8cb0d93e6931106f381d2fbd/.command.err:0.00% overall alignment rate
./5d/4bd31c8cb0d93e6931106f381d2fbd/.command.err:0.00% overall alignment rate
./5d/6ffc38f297d91e4129b4928df4b7e6/.command.err:0.03% overall alignment rate
./5d/6ffc38f297d91e4129b4928df4b7e6/.command.err:0.09% overall alignment rate
./1b/e4ad316393ef546f93cd61dac684ff/.command.err:0.00% overall alignment rate
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Hi Mike, I ran into the same issue with the key error KeyError: 'TRAV29_DV5*03'. However, adding the -m assembly flag didn't help.
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I'll second this; I'm still seeing this error with the command: Oddly enough ... this is a down-sampling study, where I'm reducing the size of the read set to see how low we can go with future sequencing ... and I only see this error at one particular fraction of the read set, not above and below. I don't think that's relevant to the error, just interesting that there'd be this "instability" in the identified alleles. |
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Hi, below is the KeyError error that I get with tracer assemble. It is only one out of 1536 samples, so otherwise the software is running very nicely. Below the error I have copied a listing of the output files that were generated; in particular the fastq files are non-empty (they are uncompressed into the files f1 and f2). I notice that the 'overall alignment rate' looks low (0.12%), but it's similar in other samples. Is this something I need to worry about? Let me know if there is a way to dig further into this,
Thanks,
Stijn
Error
Directory structure:
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