STAR/cuffdiff- BAM record error: found spliced alignment without XS attribute

Aditya Saxena

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Jun 16, 2016, 8:00:37 PM6/16/16

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Hi,

I have used STAR (v2.5.2a) to map SR-50 (stranded libraries made with illumina-TruSeq kit) reads to a mouse genome ref. I ran cufflinks (v2.2.1) on the STAR generated 'Aligned.sortedByCoord.out.bam' using the '--library-type fr-firststrand' option and merged the resulting transcripts.gtf with cuffmerge (on Galaxy).

I ran cuffdiff on my alignments like so:

cuffdiff -o MusSR50_NCBI37.2_cuffdiff_Results -p 24 -u MusSR50_MT.RU_Cuffmerge_Galaxy11.gtf -b Mus_NCBI37.2_genome.fa -L MusSR50_MT,MusSR50_RU

M1M_TrimmedAligned.sortedByCoord.out.bam,M3M_TrimmedAligned.sortedByCoord.out.bam,M3M-2_TrimmedAligned.sortedByCoord.out.bam M2R_TrimmedAligned.sortedByCoord.out.bam,M2R-2_TrimmedAligned.sortedByCoord.out.bam,M3R_TrimmedAligned.sortedByCoord.out.bam

Although the cuffdiff_Results folder has all the files with expression diff data, slurm_report for the job heads this error:-

Wed Jun 15 22:47:22 PDT 2016

You are using Cufflinks v2.2.1, which is the most recent release.

[22:47:22] Loading reference annotation and sequence.

No fasta index found for Mus_NCBI37.2_genome.fa. Rebuilding, please wait..

Fasta index rebuilt.

[22:48:09] Inspecting maps and determining fragment length distributions.

BAM record error: found spliced alignment without XS attribute

Why am I getting the BAM record error: found spliced alignment without XS attribute? Not sure if I can trust the results generated by my cuffdiff run..

Shall be grateful for your advice!

thanks in advance

Adi

PS- I ran STAR jobs like so-

STAR --runThreadN 12 --genomeDir MusIndex_NCBI37.2_STAR --readFilesIn MusSR50_STAR_MusNCBI37.2/M1M_Trimmed.fastq --chimSegmentMin 10 --outSAMtype BAM Unsorted SortedByCoordinate --quantMode TranscriptomeSAM GeneCounts --sjdbGTFfile MusIndex_NCBI37.2_STAR/Mus_NCBI37.2_genes.gtf --outFileNamePrefix MusSR50_STAR_MusNCBI37.2/M1M_Trimmed

I attach the STAR Log.out report for one of my jobs.

M1M_Trimmed_STAR_Log.rtf

Alexander Dobin

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Jun 17, 2016, 12:03:29 PM6/17/16

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Hi Adi,

I am not using cuffdiff much, but are you not supposed to use --library-type fr-firststrand for stranded libraries?

Cheers

Alex

Aditya Saxena

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Jun 17, 2016, 3:35:21 PM6/17/16

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HI Alex,

Many thnaks for the suggestion. Using -library-type fr-firststrand for my stranded libraries did the tricks. The jobs are running fine and I don't get the BAM record error: found spliced alignment without XS attribute error.