Question: HELP! My HT seq table counts are in zero
0
gravatar for j.gonzalez10
6 months ago by
j.gonzalez100 wrote:

Hello!!

I am running HT seq using my sam files (obtained from bowtie) and the gtf file I obtained from Ensemble for Phytophthora infestans. Everything is ok except that I am obtaining zero counts on my HTseq output. I have read in other post that it may be because my gtf file uses different identifiers than my sam output. But I really don't know how to fix it and it is somehow urgent! I am analyzing small RNAseq data in four different samples so I need the table counts in order to see if small RNAs are differently expressed.

Thanks!!!!

I will remain attentive!!!

Best, Juliana

ADD COMMENTlink modified 6 months ago by Jennifer Hillman Jackson23k • written 6 months ago by j.gonzalez100
1

Post the first 5-10 lines of the SAM file and the first 5-10 lines of the GTF file.

ADD REPLYlink written 6 months ago by Devon Ryan1.8k

Thanks!

First seven of the SAM file:

@HD VN:1.0 SO:unsorted

@SQ SN:NW_003302556.1 LN:4850

@SQ SN:NW_003302557.1 LN:5561

@SQ SN:NW_003302558.1 LN:4798

@SQ SN:NW_003302559.1 LN:40370

@SQ SN:NW_003302560.1 LN:4805

@SQ SN:NW_003302561.1 LN:5155

First seven of the GTF file:

supercont1.1 broads exon 10097 10114 . - . transcript_id "transcript:PITG_00002T0"; gene_id "gene:PITG_00002"; supercont1.1 broads exon 10171 10433 . - . transcript_id "transcript:PITG_00002T0"; gene_id "gene:PITG_00002"; supercont1.1 broads exon 10474 10522 . - . transcript_id "transcript:PITG_00002T0"; gene_id "gene:PITG_00002"; supercont1.1 broads CDS 10100 10114 . - 0 transcript_id "transcript:PITG_00002T0"; gene_id "gene:PITG_00002"; supercont1.1 broads CDS 10171 10433 . - 2 transcript_id "transcript:PITG_00002T0"; gene_id "gene:PITG_00002"; supercont1.1 broads CDS 10474 10522 . - 0 transcript_id "transcript:PITG_00002T0"; gene_id "gene:PITG_00002"; supercont1.1 broads exon 38775 39071 . + . transcript_id "transcript:PITG_00003T0"; gene_id "gene:PITG_00003";

ADD REPLYlink modified 6 months ago • written 6 months ago by j.gonzalez100
1

Devon's thought was right I suppose. Your BAM/SAM file was aligned to sequences named 'NW_003302556.1' etc while your GTF file contains sequences named 'supercont1.1'. These do not correspond causing HTSeq to not find reads aligned to 'supercont1.1'.

ADD REPLYlink written 6 months ago by y.hoogstrate450

Thank you very much! That was indeed the problem.

ADD REPLYlink written 6 months ago by j.gonzalez100
1
gravatar for Devon Ryan
6 months ago by
Devon Ryan1.8k
Germany
Devon Ryan1.8k wrote:

The easiest solution to this is to realign to the fasta file associated with your GTF file. Then htseq-count will be happy. While you could use samtools reheader (it's available in Galaxy) to fix this by manually converting the chromosome names, it's so easy to make a ruinous mistake there that I wouldn't advise it.

ADD COMMENTlink written 6 months ago by Devon Ryan1.8k

Thank you Devon!!! That was the most adequate solution!!!

ADD REPLYlink written 6 months ago by j.gonzalez100
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