RNA seq downstream analysis

Question

[addilynn.beach]

Hi all,

I recently completed my first RNA seq experiment. I was wondering which program would be best to use for downstream analysis of the data. The experiment was done using Illumina and Roche Nimblegen kits. The samples were enriched for a specific virus as well. Any suggestions for starting the downstream analysis for this project would be very much appreciated!

[Medhat]

What biological question you are seeking an answer to?

and as a general answer
Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks

https://en.wikibooks.org/wiki/Next_Generation_Sequencing_(NGS)/RNA

[addilynn.beach]

We are looking for long non coding RNA, but we're not sure what to expect because we haven't sequenced these types of samples before. Thank you for the links!

[Fabio Marroni]

Hi! It is difficult to help you with such a generic question. You will have more probabilities of being helped by telling us: 1) What do you mean for "downstream analysis"? 2) Did you already align the data, or do you still have to do that? 3) What was the aim of the enrichment for virus?

[addilynn.beach]

Hi, sorry for the generic question, I'm fairly new to NGS analysis. 1- I want to do a QC, align the fastq files to a genome, and create either bedgraphs or a coverage plot, and be able to call peaks from the data. 2- I have not yet aligned the fastq files. 3- The RNA used was viral, so we do an enrichment step to try to improve our alignment rates.

Answer 1

[Fabio Marroni]

1) QC, one tool that is widely used to do a generic QC is fastqc. You might want to trim reads to remove low quality end using some tool (I usually use erne-filter), and remove adapter sequences. I usually use cutadapt for that. For coverage plots you might see this post, in which several tools and options are proposed. 2) For alignment, I suggest you to see the links that medhat sent you. Hisat2 and stringtie might be a reasonable alternative. You say you have to call peaks. What peaks? Are you analysing RIP-seq data? That would change things a bit.

Answer 2

[EagleEye]

A: Any One please provide protocol for Analysing long noncoding RNA illumina NGS da