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5.7 years ago
Mamta ▴ 460

HI all,

A fasta file with precursor sequences, (IS this the hairpin file on mirBase? a fasta file with mature miRNA sequences, GOT THIS a fasta file with star sequences - Where do I get this?

Thanks!!! Mamta

miRNAseq miRNA-sequences mirdeep • 2.6k views
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5.7 years ago
A. Domingues ★ 2.5k

A fasta file with precursor sequences, (IS this the hairpin file on mirBase?

According to this source, page 6: - hairpin.fa provides the precursors, and - mature.fa the mir sequences

So, as put it hairpin = precursor sequences.

a fasta file with star sequences - Where do I get this?

In my pipeline, Mapper + MirDeep2, I did not use star sequences.

If you are using iGenomes, the Mature and Hairpin sequences are in Annotation/SmallRNA (at least for zebrafish). These sequences did cause an error to be thrown at me by mirdeep:

Error: miRNA reference this species file genomes/Danio_rerio/Ensembl/Zv9/Annotation/SmallRNA/mature.fa has not allowed whitespaces in its first identifier

I removed the whitespace from the headers with sed:

cat mature.fa | sed 's, ,_,g' > mature.noSpaces.fa ;

cat hairpin.fa | sed 's, ,_,g' > hairpin.noSpaces.fa


I did not filter for rRNA.

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Great! Thanks so much!

Did u use RFAM?

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Did u use RFAM?

No. It was very "quick and dirty" type of analysis, and I did not filter for any repeat elements. Can't help you there sorry.

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Thanks so much though! One last question I promise- I have set of normal and disease samples, can I process them using the config file at the same time. Will I get read counts for each sample as result and comparable across? Also, could you just share the results files (maybe just a few lines) with me please? I am using collaborators computer resources so do not ahve too much time to test and try.

Thanks!!!!

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5.7 years ago
A. Domingues ★ 2.5k

Will I get read counts for each sample as result and comparable across?

As far as I remember from the miRDeep2's manual yes, but I had three conditions which I ran independently in miRDeep, followed by custom analysis.

Also, could you just share the results files (maybe just a few lines) with me please?

No problem. This is the result for the wt:

#miRNA read_count precursor total seq seq(norm) dre-let-7a_MIMAT0001759_Danio_rerio_let-7a 71366.00
dre-let-7a-1_MI0001857_Danio_rerio_let-7a-1_stem-loop 71366.00
71366.00 21779.10 dre-let-7a_MIMAT0001759_Danio_rerio_let-7a 71187.00 dre-let-7a-2_MI0001858_Danio_rerio_let-7a-2_stem-loop 71187.00 71187.00 21724.47 dre-let-7a_MIMAT0001759_Danio_rerio_let-7a 71932.00
dre-let-7a-3_MI0001860_Danio_rerio_let-7a-3_stem-loop 71932.00
71932.00 21951.82 dre-let-7a_MIMAT0001759_Danio_rerio_let-7a 71121.00 dre-let-7a-4_MI0001861_Danio_rerio_let-7a-4_stem-loop 71121.00 71121.00 21704.33 dre-let-7a_MIMAT0001759_Danio_rerio_let-7a 71932.00
dre-let-7a-5_MI0001862_Danio_rerio_let-7a-5_stem-loop 71932.00
71932.00 21951.82 dre-let-7a_MIMAT0001759_Danio_rerio_let-7a 71940.00 dre-let-7a-6_MI0001863_Danio_rerio_let-7a-6_stem-loop 71940.00 71940.00 21954.27 dre-let-7b_MIMAT0001760_Danio_rerio_let-7b 9971.00 dre-let-7b_MI0001865_Danio_rerio_let-7b_stem-loop 9971.00 9971.00 3042.90 dre-let-7c_MIMAT0001761_Danio_rerio_let-7c 10510.00 dre-let-7c-1_MI0001866_Danio_rerio_let-7c-1_stem-loop 10510.00 10510.00 3207.39 dre-let-7c_MIMAT0001761_Danio_rerio_let-7c 10502.00
dre-let-7c-2_MI0001867_Danio_rerio_let-7c-2_stem-loop 10502.00
10502.00 3204.94

The explanation for each column in in the manual. Also, this is from the files named miRNAs_expressed_all_samples_*.csv. I had three of those, one for each condition. As I mentioned before, the analysis was quick and dirty and customized for this particular project.

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Thank u so much!!! This is great!

Any particular reason why u would run the conditions separately and not together?

Mamta

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It made more sense to implement it that way in our pipeline, and I wanted to make a 3-way comparison that I wasn't sure that was possible with the miRDeep. Mostly practical reasons.

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Thanks!!! :)

Mamta