Abnormal pair orientation in RNA-seq data
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5.2 years ago
michealsmith ▴ 780

Hi biostarers, please see the attached picture from RNA-seq of total RNA; that read pairs with the same orientation (RR) that span two adjacent exons. (Supposedly we should see opposite orientation LR for paired-end reads)

I know this indicates inversion for DNA sequencing; but what about in RNA-seq? The same? Or other explanation?

https://ibb.co/k0Ob5a

================ Forget that the upper blue bar is simple repeats annotation by repeatmasker. So this intron is highly repetitive, which makes me feel highly suspicious it's circular RNA here given circular RNA formation mediated by flanking repetitive sequence. But the "abnormal" read mapping caused by circular RNA usually mimicks tandem duplication, with two reads still facing opposite but outward.

RNA-Seq igv inversion • 1.8k views
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Are all your read pairs oriented this way ? What kind of library preparation and sequencing platform was used ?

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No, only small subsets are like this. Just normal standard Illumina paired-end sequencing; no strand-specificity

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Is there anything remarkable about those reads (low nucleotide diversity etc)? Have you examined the read pairs in question to ensure that they aligner is not doing anything strange.

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The read pairs are mapped onto two exons, which are unique sequence. .So mapping is perfect. However, I just put an update to post; the middle intron is highly repetitive; which makes me feel possibly the intron is doing strange things to induce structural change?

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Wouldn't this be explained by a circular RNA?

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Yes I also think about circular RNA; but circular RNA usually indicated by opposite-oriented read pairs facing outward(just like tandem duplication). I'm newcomer to circular RNA, maybe this also indicates circular RNA?

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What is the scale on the X-axis in bp?

Sometime strange things happen with experimental biology. As @Carlo said if this is an isolated example then it may be hard to come up with a logical explanation.

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Scale is around 1-2kb. I'm wondering if "strange things" indicate functional noncoding RNA, which is usually expressed in low level

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You are showing those 4 reads but there appears to be 1000x coverage in that region. They also appear to be going in the other direction as far as the coding exons go. Are the rest of the reads "normal" (FR orientation)?

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Yes the remaining are normal (FR), but the coverage here is around 100X

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Have you ckecked in circRNA databases if there are circRNAs anotated in that region?

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Yes, there are. But the read pair orientation doesn't make sense to me here that they suggests circular RNA.

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What are the sizes of these two exons, their sequences, positions of reads starts and ends within the exons and reads sequences and qualities? Are these read pairs exact duplicates? or maybe their first couple of nucleotides and start mapping positions identical?

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