Question: StringTie "missing header error" [for both HISAT2 and STAR alignment files]
1
gravatar for EagleEye
10 months ago by
EagleEye5.5k
Sweden
EagleEye5.5k wrote:

Hi,

I am getting error while using StringTie (1.3.3b) on aligned reads from both HISAT2 (2.1.0) and STAR (2.5.3a). Please help me to rectify the problem. The error is related to missing headers in the alignment files but when I print header using samtools I can see the headers in those alignment files (hisat2 and star aligned).

Steps used:

HISAT2 alignment:

hisat2 --dta --known-splicesite-infile spliceSites.txt --phred33 --qc-filter -x ht2_index/hg38 -U sample1.fastq.gz | samtools view -bS -o sample1_hisat2.bam - ;

samtools sort -o sample1_hisat2_sorted.bam -O sample1_hisat2.bam ;

samtools view -H sample1_hisat2_sorted.bam | head

@HD VN:1.0  SO:coordinate
@SQ SN:1    LN:248956422
@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

stringtie sample1_hisat2_sorted.bam -G hg38.GTF -B -e -x -C hg38.GTF -p 8 -A hg38_gene_abund.tab -o hg38_assembled.gtf

Error:

[samopen] no @SQ lines in the header.
[sam_read1] missing header? Abort!

STAR alignment:

STAR --readFilesCommand zcat --outSAMmapqUnique 255 --outSAMattributes All --outSAMattrIHstart 0 --outSAMtype BAM SortedByCoordinate --outSAMunmapped None --outFileNamePrefix path/sample1_star --runMode alignReads --genomeDir genome_dir/STAR_index/ --readFilesIn sample1.fastq.gz ;

samtools view -H sample1_starAligned.sortedByCoord.out.bam | head

@HD VN:1.4  SO:coordinate
@SQ SN:1    LN:248956422
@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

stringtie sample1_starAligned.sortedByCoord.out.bam -G hg38.GTF -B -e -x -C hg38.GTF -p 8 -A hg38_gene_abund.tab -o hg38_assembled.gtf

Error:

[samopen] no @SQ lines in the header.
[sam_read1] missing header? Abort!
ADD COMMENTlink written 10 months ago by EagleEye5.5k
3
gravatar for Russ
10 months ago by
Russ300
Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada
Russ300 wrote:

I think the issue is not in your file but in your stringtie command. It looks like it could use a bit of tweaking. The -x option requires a parameter and is interpreting the -C hg38.GTF as input, resulting in the error message. Try removing the -x (or specifying a file) and seeing if that helps.

ADD COMMENTlink modified 10 months ago • written 10 months ago by Russ300
1

Thank a lot. You saved my day. The problem was exactly what you mentioned.

  • I removed '-x' since it doesn't have any input file (unnecessary parameter)

  • I also had another problem that -C (the output file name) and the -G (reference files names) where pointing to same GTF reference file (hg38.GTF). It was replacing the original GTF reference file for the output.

ADD REPLYlink written 10 months ago by EagleEye5.5k

Moved @Russ's comment to an answer. Go ahead and accept to provide closure to this thread.

ADD REPLYlink written 10 months ago by genomax52k
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