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

all_your_base · 04-03-2013, 06:56 PM

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 · 04-03-2013, 08:53 PM

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 · 04-03-2013, 09:00 PM

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 · 04-04-2013, 05:11 AM

Quote:

Originally Posted by all_your_base

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 · 04-04-2013, 06:03 AM

Quote:

Originally Posted by all_your_base

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.

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04-03-2013, 06:56 PM	#1
all_your_base Member Location: USA Join Date: Mar 2012 Posts: 40	Six reading frame question... why all contain ORF?? 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!!

04-03-2013, 08:53 PM	#2
kcchan Senior Member Location: USA Join Date: Jul 2012 Posts: 185	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.

04-03-2013, 09:00 PM	#3
all_your_base Member Location: USA Join Date: Mar 2012 Posts: 40	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.