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Error when running “Trimmomatic flexible read trimming tool for Illumina NGS data” on RNA seq data

Hellow everyone I work with my colleagues on RNA-seq human data where we are trying to analyze these data on usegalaxy.eu. we have +50 raw single-end sequencing data files. We have started our analysis by running FastQC on all data files with no problem. Now we are trying to run "Trimmomatic flexible read trimming tool for Illumina NGS data" on the data files to cut the first 5 bases but we are getting the error in attached image for 8 of these data files. We have tried to run these 8 data files on usegalaxy.org to see if the files will run correctly but we got the same error. Hope you can help us to troubleshoot this problem.

enter image description here

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written by abdel.albsi • abdel.albsi

Instead of a screenshot you should copy/paste the error into a code box (``` before and after code). You also need to include the code you are running that is giving the error.

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written by rpolicastro rpolicastro

why a screenshot when you can just copy-n-paste the text ? save the planet.

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written by Pierre Lindenbaum Pierre Lindenbaum

1 answer

the message is quite clear. The program expects a gzipped input but your file is not.

Test this with the command line

file fastq_in.fastqsanger.gz

the output should be something like

gzip compressed data, from ...

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written by Pierre Lindenbaum Pierre Lindenbaum

Thanks for your answer. I have checked the file type and it says it is gzip compressed data. I'm running a tool called "Trimmomatic flexible read trimming tool for Illumina NGS data" on usegalaxy.org platform. I copy past the command line below

"if [ -z "$TRIMMOMATIC_JAR_PATH" ]; then export TRIMMOMATIC_JAR_PATH=$(dirname $(readlink -e $(which trimmomatic))); fi && if [ -z "$TRIMMOMATIC_ADAPTERS_PATH" ]; then export TRIMMOMATIC_ADAPTERS_PATH=$(dirname $(readlink -e $(which trimmomatic)))/adapters; fi && ln -s '/data/dnb07/galaxy_db/files/1/9/6/dataset_1966fb59-e69a-4200-8080-4d7a469d3588.dat' fastq_in.'fastqsanger.gz' && java ${_JAVA_OPTIONS:--Xmx8G} -jar $TRIMMOMATIC_JAR_PATH/trimmomatic.jar SE -threads ${GALAXY_SLOTS:-6} fastq_in.'fastqsanger.gz' fastq_out.'fastqsanger.gz' HEADCROP:5 SLIDINGWINDOW:4:20 MINLEN:40 2>&1 | tee trimmomatic.log && if [ -z "$(tail -1 trimmomatic.log | grep "Completed successfully")" ]; then echo "Trimmomatic did not finish successfully" >&2 ; exit 1 ; fi && mv fastq_out.'fastqsanger.gz' '/data/jwd/main/048/954/48954908/outputs/galaxy_dataset_f9ffd9a3-9930-496c-9619-133db44823aa.dat'

I'm getting the output below

"Picked up _JAVA_OPTIONS: -XX:MaxPermSize=2G -Xmx6G -Xms1G -Djava.io.tmpdir=/data/2/galaxy_db/tmp -Duser.home=/data/2/galaxy_db/tmp OpenJDK 64-Bit Server VM warning: Ignoring option MaxPermSize; support was removed in 8.0 OpenJDK 64-Bit Server VM warning: Ignoring option MaxPermSize; support was removed in 8.0 TrimmomaticSE: Started with arguments: -threads 6 fastq_in.fastqsanger.gz fastq_out.fastqsanger.gz HEADCROP:5 SLIDINGWINDOW:4:20 MINLEN:40 Quality encoding detected as phred33 java.util.zip.ZipException: Not in GZIP format at java.base/java.util.zip.GZIPInputStream.readHeader(GZIPInputStream.java:166) at java.base/java.util.zip.GZIPInputStream.<init>(GZIPInputStream.java:80) at java.base/java.util.zip.GZIPInputStream.<init>(GZIPInputStream.java:92) at org.usadellab.trimmomatic.util.ConcatGZIPInputStream$GZIPHelperInputStream.<init>(ConcatGZIPInputStream.java:100) at org.usadellab.trimmomatic.util.ConcatGZIPInputStream$GZIPHelperInputStream.<init>(ConcatGZIPInputStream.java:96) at org.usadellab.trimmomatic.util.ConcatGZIPInputStream.nextGzipInputStream(ConcatGZIPInputStream.java:38) at org.usadellab.trimmomatic.util.ConcatGZIPInputStream.read(ConcatGZIPInputStream.java:75) at java.base/sun.nio.cs.StreamDecoder.readBytes(StreamDecoder.java:284) at java.base/sun.nio.cs.StreamDecoder.implRead(StreamDecoder.java:326) at java.base/sun.nio.cs.StreamDecoder.read(StreamDecoder.java:178) at java.base/java.io.InputStreamReader.read(InputStreamReader.java:185) at java.base/java.io.BufferedReader.fill(BufferedReader.java:161) at java.base/java.io.BufferedReader.readLine(BufferedReader.java:326) at java.base/java.io.BufferedReader.readLine(BufferedReader.java:392) at org.usadellab.trimmomatic.fastq.FastqParser.parseOne(FastqParser.java:59) at org.usadellab.trimmomatic.fastq.FastqParser.next(FastqParser.java:179) at org.usadellab.trimmomatic.threading.ParserWorker.run(ParserWorker.java:42) at java.base/java.lang.Thread.run(Thread.java:834) Exception in thread "Thread-0" java.lang.RuntimeException: java.util.zip.ZipException: Not in GZIP format at org.usadellab.trimmomatic.threading.ParserWorker.run(ParserWorker.java:56) at java.base/java.lang.Thread.run(Thread.java:834) Caused by: java.util.zip.ZipException: Not in GZIP format at java.base/java.util.zip.GZIPInputStream.readHeader(GZIPInputStream.java:166) at java.base/java.util.zip.GZIPInputStream.<init>(GZIPInputStream.java:80) at java.base/java.util.zip.GZIPInputStream.<init>(GZIPInputStream.java:92) at org.usadellab.trimmomatic.util.ConcatGZIPInputStream$GZIPHelperInputStream.<init>(ConcatGZIPInputStream.java:100) at org.usadellab.trimmomatic.util.ConcatGZIPInputStream$GZIPHelperInputStream.<init>(ConcatGZIPInputStream.java:96) at org.usadellab.trimmomatic.util.ConcatGZIPInputStream.nextGzipInputStream(ConcatGZIPInputStream.java:38) at org.usadellab.trimmomatic.util.ConcatGZIPInputStream.read(ConcatGZIPInputStream.java:75) at java.base/sun.nio.cs.StreamDecoder.readBytes(StreamDecoder.java:284) at java.base/sun.nio.cs.StreamDecoder.implRead(StreamDecoder.java:326) at java.base/sun.nio.cs.StreamDecoder.read(StreamDecoder.java:178) at java.base/java.io.InputStreamReader.read(InputStreamReader.java:185) at java.base/java.io.BufferedReader.fill(BufferedReader.java:161) at java.base/java.io.BufferedReader.readLine(BufferedReader.java:326) at java.base/java.io.BufferedReader.readLine(BufferedReader.java:392) at org.usadellab.trimmomatic.fastq.FastqParser.parseOne(FastqParser.java:59) at org.usadellab.trimmomatic.fastq.FastqParser.next(FastqParser.java:179) at org.usadellab.trimmomatic.threading.ParserWorker.run(ParserWorker.java:42) ... 1 more Input Reads: 26986000 Surviving: 25770239 (95.49%) Dropped: 1215761 (4.51%) TrimmomaticSE: Completed successfully"

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written by abdel.albsi • abdel.albsi

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