GATK SplitNCigarReads Error

Question

[BAGeno]

Hi,

I am using GATK SplitNCigarReads for variant discovery in RNA-seq. I have reordered bam files first with picard ReOrderSam then used SplitNCigarReads for splitting and trimming of reads by using this command.

gatk SplitNCigarReads \
      -R hg19.fa \
      -I input.bam \
      -o output.bam

But I got this error.

A USER ERROR has occurred: Badly formed genome unclippedLoc: Contig null given as location, but this contig isn't present in the Fasta sequence dictionary.

Header of my file looks like this.

@HD     VN:1.6  SO:coordinate
@SQ     SN:chr1 LN:249250621    M5:1b22b98cdeb4a9304cb5d48026a85128     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr2 LN:243199373    M5:a0d9851da00400dec1098a9255ac712e     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr3 LN:198022430    M5:641e4338fa8d52a5b781bd2a2c08d3c3     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr4 LN:191154276    M5:23dccd106897542ad87d2765d28a19a1     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr5 LN:180915260    M5:0740173db9ffd264d728f32784845cd7     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr6 LN:171115067    M5:1d3a93a248d92a729ee764823acbbc6b     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr7 LN:159138663    M5:618366e953d6aaad97dbe4777c29375e     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr8 LN:146364022    M5:96f514a9929e410c6651697bded59aec     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr9 LN:141213431    M5:3e273117f15e0a400f01055d9f393768     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr10        LN:135534747    M5:988c28e000e84c26d552359af1ea2e1d     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr11        LN:135006516    M5:98c59049a2df285c76ffb1c6db8f8b96     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr12        LN:133851895    M5:51851ac0e1a115847ad36449b0015864     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr13        LN:115169878    M5:283f8d7892baa81b510a015719ca7b0b     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr14        LN:107349540    M5:98f3cae32b2a2e9524bc19813927542e     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr15        LN:102531392    M5:e5645a794a8238215b2cd77acb95a078     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr16        LN:90354753     M5:fc9b1a7b42b97a864f56b348b06095e6     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr17        LN:81195210     M5:351f64d4f4f9ddd45b35336ad97aa6de     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr18        LN:78077248     M5:b15d4b2d29dde9d3e4f93d1d0f2cbc9c     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr19        LN:59128983     M5:1aacd71f30db8e561810913e0b72636d     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr20        LN:63025520     M5:0dec9660ec1efaaf33281c0d5ea2560f     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr21        LN:48129895     M5:2979a6085bfe28e3ad6f552f361ed74d     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chr22        LN:51304566     M5:a718acaa6135fdca8357d5bfe94211dd     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chrX LN:155270560    M5:7e0e2e580297b7764e31dbc80c2540dd     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chrY LN:59373566     M5:1e86411d73e6f00a10590f976be01623     UR:file:/data/results/Projects/lifescope/hg19.fa
@SQ     SN:chrM LN:16571        M5:d2ed829b8a1628d16cbeee88e88e39eb     UR:file:/data/results/Projects/lifescope/hg19.fa
@PG     ID:hisat2       PN:hisat2       VN:2.1.0        CL:"/cvmfs/main.galaxyproject.org/deps/_conda/envs/mulled-v1-e7321ba46fa5ea4c6b9a06b78e6cd5182b33c0a47c2c86b5d610e1361f8b1686/bin/hisat2-align-s --wrapper basic-0 -p 6 -x /cvmfs/data.galaxyproject.org/managed/hisat2_index/hg19/hg19 --new-summary --summary-file summary.txt -U /tmp/19995.unp"
@PG     ID:MarkDuplicates       VN:2.20.3-SNAPSHOT      CL:MarkDuplicates INPUT=[SRX_4297650.bam] OUTPUT=/home/data/Downloads/DCM/VCF_markdublicate/SRX_4297650.bam-marked_duplicates.bam METRICS_FILE=SRX_4297650.bam.txt    MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP=50000 MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=8000 SORTING_COLLECTION_SIZE_RATIO=0.25 TAG_DUPLICATE_SET_MEMBERS=false REMOVE_SEQUENCING_DUPLICATES=false TAGGING_POLICY=DontTag CLEAR_DT=true DUPLEX_UMI=false ADD_PG_TAG_TO_READS=true REMOVE_DUPLICATES=false ASSUME_SORTED=false DUPLICATE_SCORING_STRATEGY=SUM_OF_BASE_QUALITIES PROGRAM_RECORD_ID=MarkDuplicates PROGRAM_GROUP_NAME=MarkDuplicates READ_NAME_REGEX=<optimized capture of last three ':' separated fields as numeric values> OPTICAL_DUPLICATE_PIXEL_DISTANCE=100 MAX_OPTICAL_DUPLICATE_SET_SIZE=300000 VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json USE_JDK_DEFLATER=false USE_JDK_INFLATER=false    PN:MarkDuplicates

Is there any way to resolve this error?

[igor]

Did you use the same FASTA file for alignment?

[BAGeno]

Yes I used same fasta for alignment.

Answer 1

[lucask3]

Did you solve this issue? I am having the same thing.