Hello,

I have used mirPRo software for miRNA mining and I have found my known miRNA from my 6 fastq files (3 males and 3 females, de novo fish miRNA-seq)

Now I have a "result_precursor.csv" file that could be used For known miRNA gene differential expression analysis as the input count table. (http://sourceforge.net/p/mirpro/wiki/User%20manual/)

please help me and tell the step by step script for doing DE analysis for my 2 conditions (with 3 biological replications for each). The head of the count table is as bellow:

The counts for pre-miRNAs (expression value = its mature miRNA count) in all samples | |||||||

id | E2.trimmed count | E4.trimmed count | E7.trimmed count | E3.trimmed count | E5.trimmed count | E8.trimmed count | |

hsa-let-7a-1 | 133670 | 116410 | 176291 | 204288 | 82801 | 42205 | |

hsa-let-7a-2 | 133036 | 116101 | 175640 | 203513 | 82338 | 42095 | |

hsa-let-7a-3 | 132996 | 116065 | 175571 | 203447 | 82268 | 42077 | |

hsa-let-7b | 41939 | 36542 | 42194 | 52775 | 22538 | 12063 | |

hsa-let-7c | 106191 | 122582 | 157398 | 174820 | 85421 | 32041 |

thanks

I find the DESeq2 instructions confusing, in part because the example data is far more complex, and I am not sure I can even compare just two (or more) different samples, without repeats, without treatments, conditions etc. I understand that I have to build the DESeqDataSet, but I really don't understand how to get there.from a simple table like this:

Any help for clearly a complete newbie that is not very good with R would be much appreciated!!!

Susanne

Hi Susanne,

You've likely already addressed your question by now, but DESeq2 will not work with two samples since for 2 groups (1 replicate per 'norton' and 'AMP1' group, in your example) at least 3 samples total are required, as one of the authors of DESeq2 explains in this post: https://support.bioconductor.org/p/106403/

The examples in the DESeq2 vignettes might appear complex, but they follow best practices for RNA-seq differential expression analysis, where 3 replicates per group is considered the minimum for reasonable statistical power.

Hi Susanne,

You've likely already addressed your question by now, but DESeq2 will not work with two samples since for 2 groups (1 replicate per 'norton' and 'AMP1' group, in your example) at least 3 samples total are required, as one of the authors of DESeq2 explains in this post: https://support.bioconductor.org/p/106403/

The examples in the DESeq2 vignettes might appear complex, but they follow best practices for RNA-seq differential expression analysis, where 3 replicates per group is considered the minimum for reasonable statistical power.