CollectRnaSeqMetrics shows "Sequence dictionaries differ" error

Question

[anjasta47]

Hi all! I was trying to run the CollectRnaSeqMetrics from Picard tools. My command was:

java -Xmx4g -jar picard.jar CollectRnaSeqMetrics \
I=/home/ania/STAR_results/SLX-10113.D701_D505.C80D1ANXX/Aligned.out.sam \
O=/home/ania/mappingQC/1-5.RNA_Metrics \
REF_FLAT=/home/ania/STAR/STAR-2.5.2a/bin/Linux_x86_64/gtf/refFlat.txt \
REFERENCE_SEQUENCE=/home/ania/STAR/STAR-2.5.2a/bin/Linux_x86_64/genome/GRCh38_r85.all.fa \
STRAND_SPECIFICITY=NONE \
RIBOSOMAL_INTERVALS=/home/ania/mappingQC/gencode.v19.rRNA.interval_list \
CHART_OUTPUT=/home/ania/mappingQC/file.rnaseq.pdf

I created Ribosomal_intervals file by using this script: script and making a few changes in it, namely: I changed hg19 to hg38 and gene_type to transcript_biotype

For mapping I used the STAR tool. Unfortunatelly, what I get is an error:

Exception in thread "main" picard.PicardException: Sequence dictionaries differ in /home/ania/STAR_results/SLX-10113.D701_D505.C80D1ANXX/Aligned.out.sam and /home/ania/mappingQC/gencode.v19.rRNA.interval_list

Together with many warnings like these:

WARNING 2016-07-27 11:06:36 IntervalList Ignoring interval for unknown reference: 13:60613608-60613734 + ENST00000411144

WARNING 2016-07-27 11:06:36 IntervalList Ignoring interval for unknown reference: Y:18348035-18348147 - ENST00000516858

Such warnings refere to all chromosomes.

I don't know what to do with this problem. Could you please tell me how I can solve this? I would be extremely grateful.

[GenoMax]

At least for the Sequence dictionaries differ part see the solution in this thread. It appears that you used the same script for creating intervals file so it should be applicable in your case.

[anjasta47]

I have seen this thread while looking for the solution but it doesn't solve my problem because both my sam file and intervals file are tab-delimited.

Answer 1

[Pierre Lindenbaum]

show me the BAM header for samtools view -H Aligned.out.sam and the header for gencode.v19.rRNA.interval_list

[anjasta47]

@HD     VN:1.4
@SQ     SN:10   LN:133797422
@SQ     SN:11   LN:135086622
@SQ     SN:12   LN:133275309
@SQ     SN:13   LN:114364328
@SQ     SN:14   LN:107043718
@SQ     SN:15   LN:101991189
@SQ     SN:16   LN:90338345
@SQ     SN:17   LN:83257441
@SQ     SN:18   LN:80373285
@SQ     SN:19   LN:58617616
@SQ     SN:1    LN:248956422
@SQ     SN:20   LN:64444167
@SQ     SN:21   LN:46709983
@SQ     SN:22   LN:50818468
@SQ     SN:2    LN:242193529
@SQ     SN:3    LN:198295559
@SQ     SN:4    LN:190214555
@SQ     SN:5    LN:181538259
@SQ     SN:6    LN:170805979
@SQ     SN:7    LN:159345973
@SQ     SN:8    LN:145138636
@SQ     SN:9    LN:138394717
@SQ     SN:MT   LN:16569
@SQ     SN:X    LN:156040895
@SQ     SN:Y    LN:57227415
@PG     ID:STAR PN:STAR VN:STAR_2.5.2a  CL:/home/ania/STAR/STAR-2.5.2a/bin/Linux_x86_64/STAR   --runThreadN 36   --genomeDir /home/ania/STAR/STAR-2.5.2a/bin/Linux_x86_64/genome_index   --readFilesIn /home/ania/data_FastQ/SLX-10113.D701_D505.C80D1ANXX.fq
@CO     user command line: /home/ania/STAR/STAR-2.5.2a/bin/Linux_x86_64/STAR --runThreadN 36 --genomeDir /home/ania/STAR/STAR-2.5.2a/bin/Linux_x86_64/genome_index --readFilesIn /home/ania/data_FastQ/SLX-10113.D701_D505.C80D1ANXX.fq

@SQ     SN:chr1 LN:248956422    AS:hg38
@SQ     SN:chr2 LN:242193529    AS:hg38
@SQ     SN:chr3 LN:198295559    AS:hg38
@SQ     SN:chr4 LN:190214555    AS:hg38
@SQ     SN:chr5 LN:181538259    AS:hg38
@SQ     SN:chr6 LN:170805979    AS:hg38
@SQ     SN:chr7 LN:159345973    AS:hg38
@SQ     SN:chrX LN:156040895    AS:hg38
@SQ     SN:chr8 LN:145138636    AS:hg38
@SQ     SN:chr9 LN:138394717    AS:hg38
@SQ     SN:chr11        LN:135086622    AS:hg38
@SQ     SN:chr10        LN:133797422    AS:hg38
@SQ     SN:chr12        LN:133275309    AS:hg38
@SQ     SN:chr13        LN:114364328    AS:hg38
@SQ     SN:chr14        LN:107043718    AS:hg38
@SQ     SN:chr15        LN:101991189    AS:hg38
@SQ     SN:chr16        LN:90338345     AS:hg38
@SQ     SN:chr17        LN:83257441     AS:hg38
@SQ     SN:chr18        LN:80373285     AS:hg38
@SQ     SN:chr20        LN:64444167     AS:hg38
@SQ     SN:chr19        LN:58617616     AS:hg38
@SQ     SN:chrY LN:57227415     AS:hg38
@SQ     SN:chr22        LN:50818468     AS:hg38
@SQ     SN:chr21        LN:46709983     AS:hg38
@SQ     SN:chrM LN:16569        AS:hg38

The first one is my sam file and the second one gencode.v19.rRNA.interval_list

[GenoMax]

As you can see there is a name mismatch for your chromosomes in those two files.