Question: Determination of target genes of DE miRNAs and interpretation of the sequencs?
0
gravatar for top5
7 weeks ago by
top510
top510 wrote:

Hello, I recently worked on predicting target sites of DE miRNAs by using miranda v3.3a but i get just ref sequences and alignment score etc. Then i used 'blastn' to find which genes are aligned for the ref sequences that i found in miRanda. But do you think i did right way? I do not want to do something wrong if anyone knows it could you please answer?

rna-seq sequence alignment gene • 132 views
ADD COMMENTlink modified 7 weeks ago by Kevin Blighe61k • written 7 weeks ago by top510
0
gravatar for Kevin Blighe
7 weeks ago by
Kevin Blighe61k
Kevin Blighe61k wrote:

Hi, a simpler / quicker way is likely via miRNAtap. In this example, I search for targets of [hsa]-miR-151-5p and I aggregate the findings across PicTar, DIANA, TargetScan, and miRanda. This program will rank the target genes.

require(miRNAtap)
require(miRNAtap.db)

searchTerm <- 'miR-151-5p'

mirTargetPredictions <- getPredictedTargets(
  searchTerm,
  sources = c('pictar','diana','targetscan','miranda'),
  species = 'hsa',
  method = 'geom',
  min_src = 3)

head(mirTargetPredictions)
       source_1 rank_product rank_final
27076         1            1          1
10235         2            2          2
25769         3            3          3
57572         4            4          4
171023        5            5          5
728642        6            6          6

Those rownames are Entrez IDs. We can convert these to HGNC symbols and Ensembl gene IDs:

require(biomaRt)
mart <- useMart("ENSEMBL_MART_ENSEMBL")
mart <- useDataset("hsapiens_gene_ensembl", mart)
annot <- getBM(
  mart = mart,
  attributes = c('entrezgene_id', 'hgnc_symbol', 'ensembl_gene_id'),
  uniqueRows = TRUE)

mirTargetPredictions <- data.frame(
  mirTargetPredictions,
  annot[match(rownames(mirTargetPredictions), annot$entrezgene_id),])

head(mirTargetPredictions)
       source_1 rank_product rank_final entrezgene_id hgnc_symbol
27076         1            1          1         27076       LYPD3
10235         2            2          2         10235     RASGRP2
25769         3            3          3         25769     SLC24A2
57572         4            4          4         57572       DOCK6
171023        5            5          5        171023       ASXL1
728642        6            6          6        728642      CDK11A
       ensembl_gene_id
27076  ENSG00000124466
10235  ENSG00000068831
25769  ENSG00000155886
57572  ENSG00000130158
171023 ENSG00000171456
728642 ENSG00000008128

Please explore the various parameters that are being used with these functions.

Kevin

ADD COMMENTlink written 7 weeks ago by Kevin Blighe61k

Thank you so much your answer but my species is rabbit that is why i used miranda to scan its reference genome, the package that you suggest does not include this species...

ADD REPLYlink written 6 weeks ago by top510

No problem, but, unfortunately, Oryctolagus cuniculus was not mentioned in your question.

Would it not also be a good idea to align the sequences to the annotation that already exists? - https://www.ensembl.org/Oryctolagus_cuniculus/Info/Index (see 'gene annotation' section).

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by Kevin Blighe61k

Yes i could do that but miRanda gives target sequences of genes. My main question is, after I got the target sequences, I used blastn to find out which genes these sequences belong to, is this the correct way? Or do you know another packages to do that? Because i have lack of information about it obviously...

ADD REPLYlink written 6 weeks ago by top510
1

miRanda returns the predicted target mRNA sequence of your input micro-RNAs, right?

You can use BLAST for these, but I see no reason why you cannot also use the data that is already available for this species, e.g., aligning the predicted target sequences against the gene FASTA data available at: https://www.ensembl.org/Oryctolagus_cuniculus/Info/Index

There is no major issue with your approach, provided that you understand how to filter the BLAST output for the best alignments.

ADD REPLYlink written 6 weeks ago by Kevin Blighe61k
1

Now i understand what do you mean, yes you are right i will try it now !! Thank you so much. I didn't think in the first place.

ADD REPLYlink written 6 weeks ago by top510
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 953 users visited in the last hour