Six reading frame question... why all contain ORF??

[all_your_base]

Hi,

So I have a conceptual question I'm trying to get my head around. I have some RNA-seq data and was trying to determine the ORF of each read. Of course a six reading frame translation of a given nucleotide sequence would be expected to have a significant ORF in at least one frame, as long as the sequence comes from a gene region.

However, I find short bits of sequences (my 150bp RNA-seq reads) that appears to have continuous ORFs on all 6 frames of translation without any stop codons at all... how can this be? Since there are 3 stop codons out of 64 possibilities, we should statistically see a stop every 21AA (63 bases) or so.

I realize this could be an anomaly, but this seems to be the case with about 10% of all my RNA-seq reads. I realize this could happen with repetitive sequence, but I don't think that is the case, since it is RNA-seq data.

Any thoughts or speculations are gladly welcomed!!

[kcchan]

RNA-seq libraries are almost never full length; the strands are fragmented into shorter fragments before sequencing. Therefore the reads you get are only a portion of the full mRNA. If you want to get the complete AA sequence of an RNA, you'll have to assemble your reads back together first.

[all_your_base]

Thanks for the reply. I understand this is just a small fragment of a whole mRNA, but for a span of 150 bases, I can't understand why we should find no stop codons on all 6 reading frames.

[syfo]

Since there are 3 stop codons out of 64 possibilities, we should statistically see a stop every 21AA (63 bases) or so.

...assuming the same frequency for each base, which is usually not the case. What is the GC% of this genome? Also, base distribution is not uniform and often differs between regions (gene/intergenic, exon/intron, etc). You might find GC-rich repeats in 3'UTRs for instance. Last, this subset of 10% might come from the same genomic locus.
Have you first tried fastqc on your reads?

[dpryan]

Since there are 3 stop codons out of 64 possibilities, we should statistically see a stop every 21AA (63 bases) or so.

Bases and codons aren't randomly distributed, nor should one to expect them to be.