To celebrate the release of GATK 4.0, we are giving away free credits for running the GATK4 Best Practices pipelines in FireCloud, our secure online analysis portal. It’s first come first serve, so sign up now to claim your free credits worth $250. Sponsored by Google Cloud. Learn more at

RNAseq varaint calling error

I'm following the Calling variants in RNAseq algorithm and I'm having an error message during the Split'N'Trim and reassign mapping qualities step.

The command I'm using is:

java -jar /usr/lib/jvm/java-7-oracle/jre/lib/ext/GenomeAnalysisTK-3.1-1/GenomeAnalysisTK.jar -T SplitNCigarReads -R ../hg38_V22.fa -I ./110222_SN627_0062_A81FPKABXX.1_CA_dedupped.bam -o ./gatk3/110222_SN627_0062_A81FPKABXX.1_CA_split.bam -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS

And the error message is:

WARN 12:39:54,935 RestStorageService - Error Response: PUT '/' -- ResponseCode: 403, ResponseStatus: Forbidden, Request Headers: [Content-Length: 1082, Content-MD5: eL7RUPoOe7Z5TAcnnuJyJA==, Content-Type: application/octet-stream, x-amz-meta-md5-hash: 78bed150fa0e7bb6794c07279ee27224, Date: Mon, 12 Dec 2016 14:39:53 GMT, Authorization: AWS AKIAI22FBBJ37D5X62OQ:lQXmnB3764v81X8SEvKZHnuuvD4=, User-Agent: JetS3t/0.8.1 (Linux/4.4.0-38-generic; amd64; en; JVM 1.7.0_80), Host:, Expect: 100-continue], Response Headers: [x-amz-request-id: 925E9C0064AB10AB, x-amz-id-2: habdpwVI552GHzAepL8UX//6EEwYcEsR4Xa1SiXSM/nGWQ0ldREV/zLjcv2zmCZ4Rp6wQDohikU=, Content-Type: application/xml, Transfer-Encoding: chunked, Date: Mon, 12 Dec 2016 15:35:01 GMT, Connection: close, Server: AmazonS3]
WARN 12:39:56,173 RestStorageService - Adjusted time offset in response to RequestTimeTooSkewed error. Local machine and S3 server disagree on the time by approximately 3306 seconds. Retrying connection.
INFO 12:39:56,932 GATKRunReport - Uploaded run statistics report to AWS S3

ERROR ------------------------------------------------------------------------------------------
ERROR A USER ERROR has occurred (version 3.1-1-g07a4bf8):
ERROR This means that one or more arguments or inputs in your command are incorrect.
ERROR The error message below tells you what is the problem.
ERROR If the problem is an invalid argument, please check the online documentation guide
ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
ERROR Visit our website and forum for extensive documentation and answers to
ERROR commonly asked questions
ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
ERROR MESSAGE: Couldn't write file /home/HDD3/sorted_bam_files/gatk/./gatk3/110222_SN627_0062_A81FPKABXX.1_CA_split.bam because file could not be created with exception Error opening file: /home/HDD3/sorted_bam_files/gatk/./gatk3/110222_SN627_0062_A81FPKABXX.1_CA_split.bam

Looks like there is a problem with Amazon server connection.

Am I able to fix this changing the -et argument?

Thanks in advance


Best Answer


  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie

    Hi Daniel, the Amazon server error did not cause the run failure, it's just a secondary reporting function that is set up to error out without consequence. In this case the real error is what follows; apparently a problem creating the output file named /home/HDD3/sorted_bam_files/gatk/./gatk3/110222_SN627_0062_A81FPKABXX.1_CA_split.bam

    This may indicate you do not have write permission to the output location, you ran out of storage space, or the file path is incorrect.

  • dgtiezzidgtiezzi BrazilMember

    Yer, it's working. Thanks.

Sign In or Register to comment.