miRNA analysis pipe line

Question

[Farbod]

Hi, I have sequenced 3 males and 3 females fish gonad miRNA using Illumina Hiseq.

Is there any software that we can input the raw Fastq files in it and it performs barcode removing, and clean and remove other small RNAs and then perform differential expression analysis ? it is a de novo study and my species has not any reference genome.

I want to find sexually DE miRNAs?

I have the same RNA-seq data, too. And I have used Trinity program for assembling them. Thank you in advance

[GenoMax]

In case of Illumina technology barcode/tag sequences are never part of the actual read (unless you are using some custom barcoding scheme). So you do not need to have a barcode removing step.

[Farbod]

So, is there any software same as trinity that we can use to merge males (3 replications) and female miRNA and count them (Quantify their expression same as what RSEM does) and perform DE? of course I have heard that tRNA, rRNA and other small ncRNAs must be removed manually, is there any automatic approach ?

[GenoMax]

Have you looked at: Microrna Pipeline

[Farbod]

Hi, yes, the first parts of that pipeline is not very simple and even clear for me. and it has spoken about "removing adapters". How can I find out that my fastq reads are clean or with adapters (using FastQC)? And about the last part of it, miRanalyzer, it needs a reference genome to be chosen. the same issue exist in miRDeep2, besides I do not know how to use "Rfam.fasta.gz" to remove unwanted sequences ? Do I must merge all my fastq files and then use RFAM or use them one by one (6 times in this case) and then merge males with females?

[Farbod]

Hi, would you please tell me how I must use my 3 replication of my treatments ?