# Exception : [Failed to read 8 bytes from input stream! Read 0] salmon quant was invoked improperly

I am trying to run salmon and it keeps giving me 2 java exceptions:

java.io.EOFException
at java.util.zip.GZIPInputStream.<init>(GZIPInputStream.java:79)
at java.util.zip.GZIPInputStream.<init>(GZIPInputStream.java:91)
at uk.ac.babraham.FastQC.Utilities.MultiMemberGZIPInputStream.<init>(MultiMemberGZIPInputStream.java:37)


and

java.lang.ArrayIndexOutOfBoundsException: -1
at uk.ac.babraham.FastQC.Modules.SequenceLengthDistribution.calculateDistribution(SequenceLengthDistribution.java:100)
at uk.ac.babraham.FastQC.Modules.SequenceLengthDistribution.raisesError(SequenceLengthDistribution.java:184)
at uk.ac.babraham.FastQC.Report.HTMLReportArchive.startDocument(HTMLReportArchive.java:336)
at uk.ac.babraham.FastQC.Report.HTMLReportArchive.<init>(HTMLReportArchive.java:84)
at uk.ac.babraham.FastQC.Analysis.OfflineRunner.analysisComplete(OfflineRunner.java:155)
at uk.ac.babraham.FastQC.Analysis.AnalysisRunner.run(AnalysisRunner.java:110)

at uk.ac.babraham.FastQC.Sequence.FastQFile.<init>(FastQFile.java:80)
at uk.ac.babraham.FastQC.Sequence.SequenceFactory.getSequenceFile(SequenceFactory.java:106)
at uk.ac.babraham.FastQC.Sequence.SequenceFactory.getSequenceFile(SequenceFactory.java:62)
at uk.ac.babraham.FastQC.Analysis.OfflineRunner.processFile(OfflineRunner.java:129)
at uk.ac.babraham.FastQC.Analysis.OfflineRunner.<init>(OfflineRunner.java:102)
at uk.ac.babraham.FastQC.FastQCApplication.main(FastQCApplication.java:316)


I installed salmon using Anacondas' conda install -c bioconda salmon and all other necessary packages in the same way. I thought that it could be a problem with fastqc, so I uninstalled it and then installed it manually (through the fastqc.zip file), but the output remained the same. I am using Java8:

java -version
openjdk version "1.8.0_92"
OpenJDK Runtime Environment (Zulu 8.15.0.1-linux64) (build 1.8.0_92-b15)
OpenJDK 64-Bit Server VM (Zulu 8.15.0.1-linux64) (build 25.92-b15, mixed mode)


I am running the script on a cluster with SLURM. Locally on my laptop the script is running without any issues. I am really stuck and do not know what to try, so any suggestions would be greatly appreciated.

Update Another issue that I just found is the following (the last command that salmon is trying to execute):

Exception : [Failed to read 8 bytes from input stream! Read 0]
salmon quant was invoked improperly.


Then I searched online for the issue and found the following thread:

https://github.com/COMBINE-lab/salmon/issues/129

However, updating salmon to the newest 0.9.1 version did not solve the issue.

Edit The command I am running is the following one:

python "$path/salmon_rna_seq.py" -L 'IU' -E "$path/jyvo_experiment-
metadata.yaml" -T 'gencode_mouse_m13' -C "$path/salmon_rna_seq.yaml" -I "$path_to_fastq/$name-lec_1_r1.fq.gz" "$path_to_fastq/$name- lec_1_r2.fq.gz" -O "$path/res/$name-lec_1" -S "$name-lec_1" -P '6' -X 'True'


The command is run by Pypiper: https://github.com/epigen/pypiper Inside of salmon_rna_seq.py I am also running commands to treat the data, the salmon part looks the following way:

def salmon(self):
cmd = "salmon quant -i " + self.indexed_transcriptome
cmd += " -l " + self.lib_type

if self.trim:
cmd += " -1 " + self.sample_files['r1']['fastq']['trimmed_paired']
cmd += " -2 " + self.sample_files['r2']['fastq']['trimmed_paired']
else:
cmd += " -1 " + self.sample_files['r1']['fastq']['chastity']
cmd += " -2 " + self.sample_files['r2']['fastq']['chastity']

cmd += " -o " + self.sample_dirs['salmon']['base']
cmd += " --numBootstraps=30"
self.pipe_manager.run(cmd, self.sample_files['salmon'])


Commands that the Pypiper runs are the following ones (_commands.sh Pypiper file):

zcat -c $PATH/res/old-lec_1/fastq/merged/old-lec_1_r1.fq.gz | grep -A 3 '^@.*[^:]*:N:[^:]*:' | grep -v '^\-\-$' | gzip -c > $PATH/res/old-lec_1/fastq /chastity/old-lec_1_r1.fq.gz zcat -c$PATH/res/old-lec_1/fastq/merged/old-lec_1_r2.fq.gz | grep -A 3
'^@.*[^:]*:N:[^:]*:' | grep -v '^\-\-$' | gzip -c >$PATH/res/old-lec_1/fastq
/chastity/old-lec_1_r2.fq.gz

java -jar $HOME/anaconda2/share/trimmomatic-0.36-5 /trimmomatic.jar PE -threads 6 -phred33$PATH/res/old-lec_1/fastq/chastity/old-
lec_1_r1.fq.gz $PATH/res/old-lec_1/fastq/chastity/old-lec_1_r2.fq.gz$PATH/res/old-
lec_1/fastq/trimmed/old-lec_1_r1.fq.gz $PATH/res/old-lec_1/fastq/trimmed/unpaired /old-lec_1_r1.fq.gz$PATH/res/old-lec_1/fastq/trimmed/old-lec_1_r2.fq.gz $PATH/res/old- lec_1/fastq/trimmed/unpaired/old-lec_1_r2.fq.gz ILLUMINACLIP:$HOME/anaconda2/share/trimmomatic-0.36-5/adapters
MINLEN:36

fastqc $PATH/res/old-lec_1/fastq/merged/old-lec_1_r1.fq.gz --outdir$PATH/res/old-
lec_1/fastqc/merged

fastqc $PATH/res/old-lec_1/fastq/chastity/old-lec_1_r1.fq.gz --outdir$PATH/res/old-
lec_1/fastqc/chastity

fastqc $PATH/res/old-lec_1/fastq/merged/old-lec_1_r2.fq.gz --outdir$PATH/res/old-
lec_1/fastqc/merged

fastqc $PATH/res/old-lec_1/fastq/chastity/old-lec_1_r2.fq.gz --outdir$PATH/res/old-
lec_1/fastqc/chastity

fastqc $PATH/res/old-lec_1/fastq/trimmed/old-lec_1_r1.fq.gz --outdir$PATH/res/old-
lec_1/fastqc/trimmed

fastqc $PATH/res/old-lec_1/fastq/trimmed/old-lec_1_r2.fq.gz --outdir$PATH/res/old-
lec_1/fastqc/trimmed

salmon quant -i $HOME/Genomes/mouse/salmon /gencode.vM13.transcripts.fa.sidx -l IU -1$PATH/res/old-lec_1/fastq/trimmed
/old-lec_1_r1.fq.gz -2 $PATH/res/old-lec_1/fastq/trimmed/old- lec_1_r2.fq.gz -o$PATH/res/old-lec_1/quantification --numBootstraps=30


Edit

After running manually gzip -t on a file:

-bash-4.2\$ gzip -t old-lec_1_r1.fq.gz
gzip: old-lec_1_r1.fq.gz: unexpected end of file


So, I guess the file is corrupted and the issue is with some library that is generating the file. However, reinstalling manually other libraries do not help.

• Can you run gzip -t on the file(s) to see if it/they are corrupt? That'd be the interpretation of the error messages you're seeing. Make sure to run this on the cluster, in case there's some sort of "modified in transit" networking error going on. Dec 12 '17 at 23:57
• This would be my guess. Also, however, I'm not sure how Java is involved (since Salmon is written completely in C++). Dec 13 '17 at 3:53
• It looks like FastQC is being invoked somewhere. Providing the precise commands invoked will help us troubleshoot the problem. Dec 13 '17 at 5:06
• Yes, please edit your question and show the exact commands you are using. It's hard to debug something if we don't know how to replicate it. Dec 13 '17 at 9:59
• Oh god. I swear I didn’t notice the pun in my previous comment. Dec 13 '17 at 11:11

The issue was caused by the corrupted input .fastq files that were damaged somehow upon uploading them from the local machine. We figured it out by md5sum command output comparison. We have not yet managed to actually run all of the script still, it is failing, but for another reason now.