Question

featureCounts: unable to find chromosome in SAM header

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nklier38 • 0

@017d111b

Last seen 2.7 years ago

I am using featureCounts to try and create a count table for some RNA-Seq data I collected using an Oxford Nanopore platform. I have .sam files aligned with minimap2, and am running the following command to try to get a count table:

featureCounts -a knownGene_v36.gtf -o countFile *.sam -L

Both the GTF file I am using, and the reference genome that the alignments were generated off of, are from UCSC table browser, HG38, knownGene, GENCODE v36.

Running this in the directory with the .gtf prints out an extremely long list of errors like the ones below:


ERROR: Unable to find chromosome 'hg38_knownGene_ENST00000407684.1' in the SAM header! 

ERROR: Unable to find chromosome 'hg38_knownGene_ENST00000401533.7' in the SAM header! 

ERROR: Unable to find chromosome 'hg38_knownGene_ENST00000474095.1' in the SAM header! ERROR: Unable to find chromosome 'hg38_knownGene_ENST00000506070.1' in the SAM header!

It appears to be outputting this for every gene. I originally thought there was a problem with the way the GTFs are formatted, because it's referring to the genes as chromosomes, but featureCounts seems to be pretty good about checking the gtf format before the program runs. I really don't know what I'm doing wrong- is it possibly an alignment setting that makes my SAM files improperly formatted?

RNASeq featureCounts • 1.4k views

ADD COMMENT • link updated 18 months ago by marie • 0 • written 2.7 years ago by nklier38 • 0

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Thank you for sharing! fireboy and watergirl

ADD REPLY • link 2.6 years ago garrett • 0

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I am experiencing the same issue. I first aligned the paired-end reads with bwa and then used featureCounts but I get the same error message:

ERROR: Unable to find chromosome 'NZ_CP043529_1_467' in the SAM header!
ERROR: Unable to find chromosome 'G_182_339_447_ATPG' in the SAM header!
...

But the chromosome is in my SAM header:

grep "NZ_CP043529_1_467" file2.sam 

@SQ     SN:NZ_CP043529_1_467    LN:1347
A00438:HFFWGDMXX180511:HFFWGDMXX:2:1101:18457:20964:N:0:GTCAAGTTGAGCGAGT#0      83      NZ_CP043529_1_467       346     50      91M     =203      -234    CAGTATACTCGTATATTGACAATTGCAATTTTGGTTTTCCAAGCTCCCTCATACTTGCTTAACCTTAAGATGCAAGCCGGACCATCACTTA     FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF       NM:i:0  MD:Z:91 MC:Z:3S97M      AS:i:91 XS:i:86 RG:Z:foo        XA:Z:GMGC10_047_480_898_SECY,-346,91M,1;GMGC10_055_346_782_SECY,-346,91M,4;
A00438:HFFWGDMXX180511:HFFWGDMXX:2:1101:18457:20964:N:0:GTCAAGTTGAGCGAGT#0      163     NZ_CP043529_1_467       203     50      3S97M   =346      234     CCTGAGCGTTTTCCAATGCATCTATTTTTGCATTGGGAATCATGCCATATATCTCGGCATCAATTGTAATTCAGCTTTTGGCGATTGCTGTTCCCTACTT    ,,:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF      NM:i:0  MD:Z:97 MC:Z:91M        AS:i:97 XS:i:92 RG:Z:foo  XA:Z:GMGC10_047_480_898_SECY,+203,3S97M,1;GMGC10_055_346_782_SECY,+203,3S97M,3;

and also in my GFF file

NZ_CP043529_1_467       Prodigal_v2.6.3 CDS     1       1347    72.2    +       0       ID=71508405_1;partial=10;start_type=Edge;rbs_motif=None;rbs_spacer=None;gc_cont=0.382;conf=100.00;score=72.18;cscore=68.97;sscore=3.22;rscore=0.00;uscore=3.22;tscore=0.00;

Did you find the solution?

ADD REPLY • link 18 months ago marie • 0