samtools error ([main_samview] fail to read the header from "-") during DE analysis ... empty bam file? #45
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Hi, can you provide your full input cmd? Are these cdna or direct_rna reads? |
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Thanks for getting back to me!
These are cDNA reads and I am using sbatch to start the job via slurm. See below:
#!/bin/bash
#SBATCH -J Aitken_epi2me_poly_noRNA
#SBATCH -o Aitken_epi2me_poly_noRNA.out
#SBATCH --nodes=1
#SBATCH --exclusive
#SBATCH --mem=MaxMemPerNode
#SBATCH -p emc
cd /work/epi2me
./nextflow -log /work/caitken/cea_nextflow.log run epi2me-labs/wf-transcriptomes -with-trace \
--fastq /work/caitken/data/DegronNanoporeSequencing/Poly \
--ref_genome /work/caitken/data/DegronNanoporeSequencing/sacCer3/20110902_sacCer3.fa \
--ref_annotation /work/caitken/data/DegronNanoporeSequencing/sacCer3/20110902_sacCer3.gff \
--de_analysis \
--sample_sheet /work/caitken/BarcodesPoly.csv \
--out_dir /work/caitken/data/DegronNanoporeSequencing/outputPoly_noRNA \
-c /work/caitken/data/DegronNanoporeSequencing/my_config.cfg
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
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… On Nov 16, 2023, at 7:04 AM, Sarah Griffiths ***@***.***> wrote:
Hi, can you provide your full input cmd? Are these cdna or direct_rna reads?
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Hi @cea295933 What is the exit code you are getting from that process that has the error You can actually delete this section from any config you have |
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Thanks for taking the time to look into this!
The error code I see is:
3496 Command executed:
3497
3498 minimap2 -t 4 -ax splice -uf -p 1.0 "genome_index.mmi" "seqs.fastq.gz" | samtools view -Sb > "output.bam"
3499 samtools sort -@ 4 "output.bam" -o "WTpoly2_reads_aln_sorted.bam"
3500
3501 Command exit status:
3502 1
3503
3504 Command output:
3505 (empty)
3506
3507 Command error:
3508 [WARNING] Indexing parameters (-k, -w or -H) overridden by parameters used in the prebuilt index.
3509 [main_samview] fail to read the header from "-".
3510
3511 Work dir:
3512 /work/epi2me/work/85/754a2fa2bd94abf6deaf62d3491903
The contents of my config file are:
1 executor {
2 $local {
3 cpus = 64
4 memory = "256 GB"
5 }
6 }
Our HPC has 512 GB and 128 CPUs available per node. It looks to me like the sbatch code is overriding the config file, because squeue returns 128 CPUs running for the job. But I will try running this with the added code and will amend to config file auto to request all the available CPUs and memory (unless you suggest I try just shy of the max memory, say 480 GB)
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
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… On Nov 16, 2023, at 10:42 AM, Sarah Griffiths ***@***.***> wrote:
Hi @cea295933 <https://github.com/cea295933>
What is the exit code you are getting from that process that has the error fail to read the header from "-". This step is very memory intensive process and I have seen this same error message/empty bam appear if the alignment process has run out of memory. Ensure the global config has a high memory set and consider adding --minimap2_index_opts to eg. -w 25 or -w 50
You can actually delete this section from any config you have
executor {
$local {
cpus = 4
memory = "8 GB"
}
}
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Sorry I see you have supplied the full input cmd above, what is the reference annotation and reference genome you are using if publicly available? |
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Also how many samples are you running? |
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I am running two replicates of each sample … I know 3 or more is best but elsewhere on the GitHub I see that 2 is ok (and know that DESeq2, for example, can accept just 2 replicates. This is a historical set of samples that we used for Illumina sequencing and so don’t have more replicates. I’m trying to compare our results here to what we obtained previously using DESeq2 on those Illumina runs to determine whether to purchase a PromethION instrument.
We actually have 8 samples total, but I am running them in two batches. They represent either total RNA or a fraction of Toal RNA (associated with ribosomes). We have two replicates for each condition (WT/Mut and Total/Poly). All I really need is a read counts so that I can go into DESeq2 to repeat our previous analysis and compare. I’m certainly interested in the DE numbers the workflow provides, but I also want to determine DE for the (Poly/Total) reads, when comparing mutant to WT, and I know how to setup DESeq2 to achieve that using the R package directly.
Thanks!
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
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… On Nov 16, 2023, at 12:13 PM, Sarah Griffiths ***@***.***> wrote:
Also how many samples are you running?
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If you have a reference transcriptome available and supply it to the ref_transcriptome parameter does the workflow progress past this part of the workflow? Trying to work out if the reads or something to do with the string tie merged transcriptome we are using could be causing this error. |
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If I supply a reference guided transcritptome, it crashes at the salmon step … I get an error about needing to upgrade salmon (described in issue 43 but also pasted below):
Process pipeline:differential_expression:count_transcripts (1) terminated with an error exit status (1)
Command executed:
salmon quant --noErrorModel -p "4" -t "ammended.ref_transcriptome" -l SF -a "WTpoly1_reads_aln_sorted.bam" -o counts
mv counts/quant.sf "WTpoly1.transcript_counts.tsv"
seqkit bam "WTpoly1_reads_aln_sorted.bam" 2> "WTpoly1.seqkit.stats"
Command exit status:
1
Command output:
(empty)
Command error:
Version Info: ### PLEASE UPGRADE SALMON ###
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
***@***.*** ***@***.***>
845.437.7430
… On Nov 16, 2023, at 12:25 PM, Sarah Griffiths ***@***.***> wrote:
If you have a reference transcriptome available and supply it to the ref_transcriptome parameter does the workflow progress past this part of the workflow? Trying to work out if the reads or something to do with the string tie merged transcriptome we are using could be causing this error.
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Could you try decreasing the memory directive to something like 64GB? Is there any further message in the salmon error. I think the part about updating the version isn't what's causing it to break - As we have that message in our automated tests as well which don't fail. |
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A few questions:
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Here's what I get if I run DE only a la GitHub test code (DE only test code from GitHub runs fine): 613 ERROR ~ Error executing process > 'pipeline:differential_expression:count_transcripts (1)' |
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If I run without DE, no issues with these data or with test data. |
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One issue I can see with running with supplied transcriptome is that there are no gold-standard (SGD, RefSeq, NCBI, etc.) yeast transcriptomes that contain good UTR info. In fact, most (if not all) are just ORFs, and just one isoform per gene. There are studies that have tried to investigate this(Pelechano, et al. for example) but they do not report a curated transcriptome and these have not been adopted by the big databases. |
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Just to clarify:
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Test DE analysis runs fine. Log below: Last login: Wed Nov 15 19:06:08 on console The default interactive shell is now zsh. Welcome to Ubuntu 20.04.6 LTS (GNU/Linux 5.4.0-153-generic x86_64) System information as of Thu 16 Nov 2023 12:29:58 PM EST System load: 0.0 Users logged in: 3 24014 [ee/9bb70f] process > pipeline:differential_expre... [100%] 1 of 1 ✔ |
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ok and now try reference guided |
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running all three now ... btw, the --sampel_sheet test_data/sample_sheet.csv gave me problems previously ... I don't remember if it's not included in the test data one downloads, or whether it's in a different directory or what ... I ended up having to create my own sample sheet (or move it to the directory ... can't remember) ... perhaps you could look into that separately... it might be giving others trouble too |
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good idea, yes will add it to the downloadable test set |
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two jobs with my data appear to be running stably, and test run ran without issues (see below). when I get samtools error, it is after long stable run (over 1 h). whereas when I get salmon error, it's usually after 10 minutes of run or less. Both running now for at least 10 m N E X T F L O W ~ version 23.10.0 WARN: Found unexpected parameters:
|||||||||| _____ ____ ___ ____ __ __ _____ _ _
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circle back once those have completed? Thanks so much for all of your help! |
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No worries, sorry it's not working! For the salmon breakage one - if you go in to a work directory for the map_transcriptome process do the bams contain alignments? I was able to recreate the error you had in the other ticket by inputting an empty bam to that step. That's an error we should be catching and reporting to the user. Perhaps if you run the workflow with your inputs without the --de_analysis parameter you could share the output report wf-transcriptome-report.html just to check there are alignments between the reference_genome and your reads. |
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BAM file doesn’t look empty … scp to my local machine is 40% at 762 MB transferred
du -sh only show 4.0K, however.
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
***@***.*** ***@***.***>
845.437.7430
… On Nov 16, 2023, at 1:27 PM, Sarah Griffiths ***@***.***> wrote:
No worries, sorry it's not working! For the salmon breakage one - if you go in to a work directory for the map_transcriptome process do the bams contain alignments? I was able to recreate the error you had in the other ticket by inputting an empty bam to that step. That's an error we should be catching and reporting to the user. Perhaps if you run the workflow with your inputs without the --de_analysis parameter you could share the output report wf-transcriptome-report.html just to check there are alignments between the reference_genome and your reads.
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tried to send .html reports but I can't attach in GitHub |
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I think if you zip them then you can attach |
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I have a meeting until later this afternoon ... so not ignoring if I'm radio silent. Will send along results of current runs once I have them. I also added a third run with the reference-guided (with our data) as a point of comparison |
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Could you go to the work directory of the merge_transcriptomes and if its small enough perhaps zip up and send the final_non_redundant_transcriptome.fasta and if possible also the gtf file from that directory? Just to check it looks sensible? |
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Files attached below. Some updates on where things stand here (updating my posts above). I've been chasing the memory constraint lead. I realized that the jobs I'm running on our HPC our running entirely on virtual memory and using almost no RSS. So for example, the results of htop on the running node show almost every process running at 48 GB of virtual memory and using only 0.4% of available memory (I can provide a screen grab of that if you want). In trying to chase this down, I realized that the memory limits within SLURM were not set properly. Unfortunately, correcting those (I can now ask SLRUM to give me all 512 GB of RAM on the node and it will) still result in the same odd virtual memory usage (unless this is actually normal?) and the processes still fail in the same way. I also noticed that it seemed like nextflow/docker were ignoring my config file that I was passing (I still saw what looked like 4 CPU and 8 GB of ram ... the default ... in the nextflow log). So I modified nextflow.log just to change those two lines to match what I'm requesting (128 CPU and 512 GB of RAM). But still the same failure. So that brings me to my question: **Is this virtual memory usage, and the very low physical memory usage expected? If not, do you have ideas of where to look? I'd like to make as much progress as possible investigating solutions on our end, as I know you're very busy! ** |
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ok those files look reasonable. Its only a very small transcriptome so although you have a lot of reads, I wouldn't expect this step to require that much memory. Could you possibly try to run the workflow on a laptop with docker or singularity? You could even try the desktop app EPI2ME https://labs.epi2me.io/downloads/. Asking my team if they have any ideas about your SLURM issues. |
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I will try … I have tried previously to run in on a PC, but it takes absolutely forever. I was able to get it to run on one barcode, but it took 44 hours. I then tried with everything together, but I got errors I had not seen previously. I have also tried running it on my Mac (Mac Studio), as it’s relatively powerful. But the desktop app does not play well with the new apple silicon processors … would that be any different if I run through the terminal?
I will try running with this smaller dataset on our PC and let you know what I see.
Thanks for sticking with me!
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
***@***.*** ***@***.***>
845.437.7430
… On Nov 20, 2023, at 9:25 AM, Sarah Griffiths ***@***.***> wrote:
ok those files look reasonable. Its only a very small transcriptome so although you have a lot of reads, I wouldn't expect this step to require that much memory. Could you possibly try to run the workflow on a laptop with docker or singularity? You could even try the desktop app EPI2ME https://labs.epi2me.io/downloads/. Asking my team if they have any ideas about your SLURM issues.
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ok ... I just did this with a further subsetted dataset (only 10 fastq.gz files per barcode). Ran on a local PC using desktop GUI ... received the exact same error (log below). I ran this further subsetted version on our HPC and again, same error. I then tried on the local PC again, but using different genome files (I had previously tested this on our HPC but that was before I realized I should not provide transcriptome when using DE and instead rely on generated transcriptome). The good news: it looks like it proceeds past the point where we've seen the error previously. A few questions/ideas:
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just check on our HPC and the same holds true there ... it runs further with genbank files but crashes at the pipeline:differential_expression:deAnalysis(1) step (see below) 2189 [b6/d0517e] process > output (16) [ 69%] 16 of 23 |
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Ah no I will update that about the reference transcriptome requirement. Oh that's good, we have only tested it as mentioned in the documentation with annotation files from Encode, Ensembl and NCBI. I think just ensure the strand column in your ref annotation file only has + or - or i think . is ok as well. Let me know if you need any guidance on fixing it, feel free to send the ref_annotation you are using or a link to where its from and I can check it. No it should be ok to set that it needs to be present in 2 samples. One thing to try may be to reduce the threads parameter to one or two, which will make some steps take a bit longer but may reduce memory requirement at the transcriptome alignment step. Also in the output directory execution folder did you have any example report.html and timeline.htmls, do they have any info about memory consumption before the workflow failure? |
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Attaching the ones I tried that work ... one of these is NCBI_RefSeq so should work .... but I see "." for entries like centromeres and telomeres ... suggestions on how to fix (I don't remember my Perl for text editing all that well)? Or alternatives for yeast? |
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Here is the execution folder. Bear in mind this is the heavily downsampled data set (only 10 files per barcode), it's likely not testing memory limits like the others. That said, it still is eating up 80 GB of virtual memory on the HPC ... and only 1 G RAM ... is that normal? I will say that for the other jobs, it was only the preprocessing reads step that appeared hugely memory intensive, at least according to these reports. And that always worked fine on the HPC (whereas it takes ages on a local PC) |
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I think any "." entries are ok, have you tried the genomic.gtf as the annotation file from the genbank zip folder, Oh I also noticed |
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ok ... trying both of these even now, I can find the "counts.tsv" in the working folder ... is that complete at this point of the pipeline? if so, I can use that to run DESeq2 in R myself ... (at least as long as this now works on the complete dataset ... haven't tried that yet) |
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those don't appear to work ... attaching the files in the work directory in the event they spark any ideas |
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Hi, hmm looks like you have all novel transcripts - does BK006934.2 match up with the reference file fasta header? |
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oh sorry i see you transcript_ids are all 'unassigned_transcript_1234' is that expected? |
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yeah, that's strange. on one of my previous runs I didn't see that ... I don't remember now whether it was refseq or genbank (or it could be a gff vs. gtf) .... I'll try to figure what the combination was, but that all_counts.tsv is below |
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ok ... if I run using the GenBank .fa and .gff, I get transcripts assigned. But if I replace with the .gtf (or use any combination of the RefSeq files), I get either unassigned transcripts or MSTRG (which I think come from stringtie). But I appear to get the same/similar error for all. NCBI Gengank .fa and .gff Error NCBI Genbank .fa and .gtf Error NCBI Refseq .fa and .gff Error NCBI Refseq .fa and .gtf Error |
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Sorry, I feel like its nearly there! Does it retry that DE_analysis step? I would expect that error but it to retry that step 4 times and hopefully one of the retries will work, as we iterate through trying a few different gtf2/gff3 R options. Check you are on the latest version within the app (if you are in the app). Or try |
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I think so too … thanks to you!
It looks like it retries twice but fails at that moment:
2379 [27/4f170f] process > output (13) [ 69%] 16 of 23
2380 Retry deAnalysis with gff format setting and version removal.
2381 Retry deAnalysis with gtf format setting and version removal.
2382 [58/b882f6] NOTE: Process `pipeline:differential_expression:deAnalysis (1)` terminated with an error exit status (1) -- Execution is retried (2)
2383 [a4/ff1720] NOTE: Process `pipeline:differential_expression:deAnalysis (1)` terminated with an error exit status (1) -- Execution is retried (3)
2384 ERROR ~ Error executing process > 'pipeline:differential_expression:deAnalysis (1)'
I tried the update but received the below error. Perhaps I need to ask my sysadmin to run it? Is it ok to run this on the head node or do we need to run it on the compute nodes?
***@***.***:/work/epi2me[07:47]$ ./nextflow pull epi2me-labs/wf-transcriptomes
Checking epi2me-labs/wf-transcriptomes ...
epi2me-labs/wf-transcriptomes contains uncommitted changes -- cannot pull from repository
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
***@***.*** ***@***.***>
845.437.7430
… On Nov 21, 2023, at 4:17 AM, Sarah Griffiths ***@***.***> wrote:
nextflow pull epi2me-labs/wf-transcriptomes
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No i'm sorry its still not working! Ok on the files in your files.zip I did this to find the two which have strand set to . , Which is causing this error This is an error being created by stringtie I've not seen it before but will add a step to remove any non stranded annotations hopefully will release that by end of the week, in the meantime you could add these lines to line 92 of |
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Above and beyond! … where can I find the subworkflows directory? I tried find, but it’s taking a while ...
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
***@***.*** ***@***.***>
845.437.7430
… On Nov 21, 2023, at 12:45 PM, Sarah Griffiths ***@***.***> wrote:
No i'm sorry its still not working!
Ok on the files in your files.zip I did this to find the two which have strand set to . ,
grep -P '\t\.\t\.\t' annotation.gtf
BK006938.2 StringTie transcript 1302119 1302626 1000 . . gene_id "MSTRG.844"; transcript_id "MSTRG.844.1";
BK006938.2 StringTie exon 1302119 1302626 1000 . . gene_id "MSTRG.844"; transcript_id "MSTRG.844.1"; exon_number "1";
Which is causing this error values in 'transcripts$tx_strand' must be "+" or "-", If I run the downstream DE_analysis without these lines it seems to complete.
This is an error being created by stringtie I've not seen it before but will add a step to remove any non stranded annotations hopefully will release that by end of the week, in the meantime you could add these lines to line 92 of subworkflows/differential_expression.nf which might fix it
grep -v -P '\t\.\t\.\t' annotation.gtf > tmp.gtf
mv tmp.gtf annotation.gtf
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ok .. I think I found it. Before line 92, right? I see the de_analysis.R script being called on line 92, and it appears to use the annotation.gtf as an input if I understand if correctly |
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ok, I changed differential_expression.nf as below: """ but now I get the below the moment I launch the workflow, so I think I made the edit incorrectly (sorry!) ERROR ~ Module compilation error
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ok sorry forgot to escape the '\' so should of been - |
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completed (with the downsampled data set)!!! Huzzah! I just launched jobs for the full data set to see if those work. Thank you!!! |
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ah okay yes, now the challenge is the full data set - let me know how it goes |
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completed! Thank you so much. One question: is there a meaningful difference in the pipeline with and without the minimap2 option we incorporated ("w 50")? Trying to determine whether it's worth testing it without that flag or not, now that everything works. |
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Good point! It may result in a few more unaligned reads, so you may be missing some transcripts. Now its working it might be worth trying with the standard "w 10" to see if it still works, or "w 25". |
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Closing through lack of response and issues solves |
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My apologies for not closing the loop here. Yes, this is now resolved. Running smoothly on the full dataset and with either set of minimap2 options. Thank you for all of your help!
Colin Echeverría Aitken
Assistant Professor
Biology Department
Biochemistry Program
Vassar College
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845.437.7430
… On Dec 12, 2023, at 5:57 AM, Sarah Griffiths ***@***.***> wrote:
Closing through lack of response and issues solves
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Ask away!
Hello,
My workflow seems to be failing as a result of what I think is a samtools error.
Command:
minimap2 -t 4 -ax splice -uf -p 1.0 "genome_index.mmi" "seqs.fastq.gz" | samtools view -Sb > "output.bam"
3499 samtools sort -@ 4 "output.bam" -o "WTpoly2_reads_aln_sorted.bam"
Error:
[WARNING] Indexing parameters (-k, -w or -H) overridden by parameters used in the prebuilt index.
[main_samview] fail to read the header from "-".
I've taken a quick look at the files being maniupulated here (in the offending work directory). The .mmi does not appear to be human readable but is at least not empty (I can provide if you like ... GitHub won't allow me to upload). And seqs.fastq.gz is certainly not empty: 5G compressed and 20G uncompressed (I am pasting just the first entry for your reference below). However, output.bam is empty, and so I think this causes the issue for the samtools sort command. Any idea what could be causing this?
@4aabeb37-0ae5-4289-ab27-e6f246a7e21a runid=215283ee2a07610bc86fc4baad99de671b475048 read=74966 ch=2198 start_time=2023-09-23T00:45:55.980051-04:00 flow_cell_id=PAO05278 protocol_group_id=Vassar_smm092223 sample_id=NB_A11-H11 barcode=barcode84 barcode_alias=barcode84 parent_read_id=4aabeb37-0ae5-4289-ab27-e6f246a7e21a basecall_model_version_id=dna_r10.4.1_e8.2_5khz_400bps_hac@v4.2.0
ATGTTATGTCCTGTACTTCGTTCAGTTACGTATTGCTAAGGTTAAGTAGTGGACCTAGAACCTGTGCCACAGCACCTCGCCTACCGTGACAAGAAAGTTGTCGGTGTCTTTGTGACTGCCTGTCGCTCTATCTTCAGAGGAGAGTCCGCCGCCCGCAAGTTTTTTTTTTTTTTTTTTTTTTTTTTGCCGATGTGATTCTAAAGATGTCATTCATATATTCAAGTTAAAAAATATATAGAATTGATAAGCCATATATAGAAACAATTACTAAAGAGAGAAAAAAAACCAAGTCTGGTAAAAGAAAAAGAAAAAGAGCCTAATATACGACACTTTGGTTCAAATAATTCACCAGAAGTCCTACGTTTTATCCTCCACTTATTATTATATCATCTTGTTAATTTTATTTCTTTAAAATTTAGCAAATTGCTTGTTGGCCTTACCGGCAGCAGCAGAGACCTTGACGACGTTGAATCTAACAGTCTTGGAGATTGGTCTACATTGACCAACGGTAACAATGTCACCAACTTGGACACGGAAAGCTGGGGAGACGTGGACTGGGACGTTCTTGTGTCTCTTTTCGTATCTGTTGTACTTTGGAATGTAATGCAAGTAAGCTCTTTCTGATGACAATGGTACGGTGCATCTTGGTGGAGACGACGGTACCGGTCAAGATCTTACCACGGATGGAAACTAAACCAGTGAATGGACATTTCTTATCAATGTAAGAACCTTCAATAGCGGTCTTTGGGGTCTTGAATCCCAAACCGGCATTCTTATACCATCTCTTGGTTCTCTTGGAAGTCTTGACCTTTGGATTGTTGAAGATGTGAGGTTGCTTTTGGAAAGCTCTTTAGATTGAACAGTTAATTCAGTGGACATCTTTTTTCCCTGGCTTGATACCCCAAAGTCCAAGGGCAATTCGAACTGGAAAGCAATATCAGCACCAACAGAAACACAAAGACACCGACAACTTTCTTGTCACC
+
%%&'*(++)%%(+,**++++235891122<><=>>FIDDCCFD=714211<BA?BBHGITFFEDDEEEE{HIPCBA@@?@ADJPHH{I{NHE=<<=BCECEIDGHGNHFJHI{{JHPFDICE{F@@AAFBCCBI=888>KHFNL{HJEGDDDEC=EHEHJ@D67>?ADRI{{{{{{{{{HBFCIJB?=.,,9{>:9<433267998:99:?>E{J{P{;:99@@b<>?DHME=>AAG>???C@?777;>@bw>@?AFQEJSIJHIHFGH{F{QIGEH{{FCCBB^PFHQJJFDIJKLHEGGHKF{{KGDJS{K{GFF{H{OO{I{KKN{HJIKHRMJ{DEJFFDFCBEIGHJG{GFNEEKEHEKLHDDB@==;:=DCABCDBCEEIIEEABBGKGUEHHJHKJHGABC@BC5546==>=F6666B@BFJFFDDCEGJEC5;;IFB==?A=76689FEGD@A@AIKGGMFEG@{9=<AC=BKFEGHGCDE{=444--,,02+**'('''+-/CDEGIHCA?@eee{HEFHE{GJ{IF{HPDFF@CA889=B?@coc;988,012677452003404476,***00+((/)6<;=::>@FFDEFBFEDHEIG{{{H6666@C.,,,<;FEGDC=77BBFGI8889FEDEDFCBCDEG<;@dlph@??GG:9:YI{JFFBE7(((788:BBFEFFFDKJE{LHI{GBDDF{@@>BDAD?>/../1011.-,-33=>@EELGJEG{HIHIFFBB@ALLGGKFHFFEDHGFHGGIJEDCHCJDEIGFJGHUIFXGHHFEIIOGMFFIGGCC:94&
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