Small RNA annotations

[sanatbhadsavle]

Hi, I have a few questions about the annotations provided by TEsmall. The reads that are annotated as tRNA, are they tRFs? Or are they part of tRNA anticodons or both? The reads that are annotated as Sense_TE and anti_TE, do they represent RNA species transcribed from transposons? Or are they RNA species that target said transposons? Thanks in advance for your help.

[olivertam]

Hi,

Thank you for your interest in the software. If you are looking at the count_summary.txt, the reads annotated to the tRNA are corresponding to the tRNA anticodons. If the tRF were annotated as TE, they will be assigned to the TE annotation instead. If you are interested in the tRFs that were annotated as TE, they should be in the xxxx.3trf.TE.mapper.anno Regarding sense_TE and anti_TE, the former are reads that are in the same orientation as the TE, and could imply those transcribed from them. The latter are in the opposite orientation to the TE, and thus "could" target the said transposons.

Thanks.

[sanatbhadsavle]

Thanks Oliver, That helps. I did look through my xxxx.3trf.TE.mapper.anno file which had lots of annotations for sense and anti TE. I read in the TESmall Frontiers paper that you found a subset of these reads tRFs from a specific tRNA arm. I was wondering how you did that analysis. How would I a go about figuring out which antisense TE is a tRF and then further figuring out the which tRNA anticodon it derives from. I also get a few a differentially expressed reads that are annotated as introns and exons. Are the intronic reads generally considered as intronic miRNAs? Similar to the Vimentin miRtron you see in the Frontiers paper. I am just trying to figure out the biological relevance of these introns and exons that seem to be differentially expressed. Thank you for all your help.

[olivertam]

Hi,

From what I understand, tRF contains a CCA tail on the end that is not genomically encoded, and thus the "standard" alignment strategy of not allowing mismatches will miss those. Thus, the algorithm takes things that are unmapped, look for those with "CCA" at the end (thus potentially tRF), trims them, and then remap and annotate them. Looking back at the Frontiers paper, it appears that they were mapping the short reads to various tRNA consensus, and assessing which tRNA shows differential expression corresponding to the TE-targeting tRFs. That might be a good way to start. Regarding intronic and exonic reads, they are typically not annotated (by miRbase in our case) as miRNA. These are reads that are coming from regions that are designated gene (protein-coding mostly), so genic introns and exons. They "could" be miRtron, but unless they have been previously characterized, it's hard to know what they are exactly. You would either need to look through the literature to see if these exonic/intronic sequences have been reported as miRtron, or perform analysis similar to that which uncovered Vim miRtron to know for sure. Hope that is helpful.

Thanks.

[sanatbhadsavle]

Hey, I looked at my .3trf.TE.mapper.anno file and it looks like this (attached) I am confused as to how I would identify tRFs from this. I apologise for badgering you with questions, I am relatively new at this.

On Thu, Aug 25, 2022 at 1:51 PM Oliver Tam @.***> wrote:

Hi, From what I understand, tRF contains a CCA tail on the end that is not genomically encoded, and thus the "standard" alignment strategy of not allowing mismatches will miss those. Thus, the algorithm takes things that are unmapped, look ZjQcmQRYFpfptBannerStart This Message Is From an External Sender This message came from outside your organization.

ZjQcmQRYFpfptBannerEnd

Hi,

From what I understand, tRF contains a CCA tail on the end that is not genomically encoded, and thus the "standard" alignment strategy of not allowing mismatches will miss those. Thus, the algorithm takes things that are unmapped, look for those with "CCA" at the end (thus potentially tRF), trims them, and then remap and annotate them. You should be able to determine which antisense TE is a tRF based on the tRF annotation, but depending on how processed that read, it might be tough to identify the exact tRNA anticodon it derives from. Regarding intronic and exonic reads, they are typically not annotated (by miRbase in our case) as miRNA. These are reads that are coming from regions that are designated gene (protein-coding mostly), so genic introns and exons. They "could" be miRtron, but unless they have been previously characterized, it's hard to know what they are exactly. You would either need to look through the literature to see if these exonic/intronic sequences have been reported as miRtron, or perform analysis similar to that which uncovered Vim miRtron to know for sure. Hope that is helpful.

Thanks.

— Reply to this email directly, view it on GitHub https://urldefense.com/v3/__https://github.com/mhammell-laboratory/TEsmall/issues/14*issuecomment-1227639810__;Iw!!KwNVnqRv!H6CWp-68pv77DoF8EBjZMEjNK-OF6z4fgvI86jxEKKOnf6kJMRik8eiIWNe-rYRZA0W7f7-hPCFs9FT10-FMBSIpElR7dA$, or unsubscribe https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/AWLQFNR7U6FQ6UMU5JDSM2TV266BJANCNFSM57P6VVPQ__;!!KwNVnqRv!H6CWp-68pv77DoF8EBjZMEjNK-OF6z4fgvI86jxEKKOnf6kJMRik8eiIWNe-rYRZA0W7f7-hPCFs9FT10-FMBSLWKcmpww$ . You are receiving this because you authored the thread.Message ID: @.***>

-- With regards Sanat Bhadsavle

[olivertam]

Hi,

I would probably start by looking for differentially expressed sRNA from anti-TE and tRNA first, and get a list of potential tRFs that could be misregulated in your experiment. You can then select the tRFs that match either the anti-TE or tRNA, and determine if they make up a significant portion of your sRNA pool. You can then try to align it to your tRNA consensus to see if there is a strong pileup at the 3' end. In essence, I think that's what was done in the TEsmall paper. Hope that gives you a head start.

Thanks.