Mapping /Need software

Question

[Arash]

Hi,

I want to do mapping of my trimmed RNA seq reads with gallus gallus reference gene.I have some questions:

1- Which program is more suitable for mapping (Bowtie2 ; Tophat or HISAT2)?Is any differences among them? or they are same?

2- I want to repeat my previous question : which type of gallus gallus reference have to be use? annotated gene ?which format? .gtf?please explain it for me

Sorry for my questions Thanks

Answer 1

[Jeffin Rockey]

1) I would recommend STAR (link)for the mapping of RNA Seq data if your machine has sufficient RAM.Else go for tophat or so.

2) For reference, you may download toplevel fasta and gtf from ensembl or so. To get those, go to ensembl,choose gallus gallus for organism.Then you should be able to see links to download fasta and gtf etc.

You will have nucleotide sequences of chromosomes/scaffolds in the fasta file whereas in the gtf you have coordinates and details of exons,transcripts,genes etc.

[Arash]

Hi,Thanks.. I visited the ensembl (Chicken) .I find the FASTA file but they contains all chromosomes separately. I have to download separately? and i also , found genes, cDNAs, ncRNA, proteins!!! Is it possible for you that put for me the correct links for ref in both formats?

Thanks A.G

[GenoMax]

You will find a single file with all chromosomes here at Ensembl.

[Antonio R. Franco]

what are you intending to do ?. An RNA-Seq analysis ?. Looking for variants ? If an RNA-Seq is intended, maybe you can give a look to Kallisto and Sleuth to do the mapping and the DE analysis Why? For simplicity, because you need fewer computer resources and because they are rapid and efficient

[Arash]

Hi, I want to do RNA seq analysis to compare the case control for expression analysis

[Antonio R. Franco]

Go ahead. Use Kallisto o Salmon to run the mapping step. You need to get a file with the coding sequence to run it. The Kallisto command line can be written by a two-year-old boy because of its simplicity. Then, you have several options to run the differential expression analysis. You can use sleuth under R, or use DESeq2, limma, etc according to the instructions included in the corresponding vignettes. There is an R package to convert the kallisto format to the regular format used for the "traditional" packages But if you read the papers, you will be happy with the information that sleuth provide, and it need only a few code lines, There are tutorials available on the internet to learn how to use them