Refrence for transcriptiome analysis

[abpatel2288]

Should i use DNA ref or RNA ref. for mapping using gs reference mapper ??

What data base set should be used for finding expression RPKM, from transcriptome reads of cow?

Sequencing was carried by 454 gs-flx.

[jmartin127]

Since the reads you get from 454 will be DNA (the cDNA is sequenced), then you should align to a DNA reference. With 454 you may not have enough sequencing depth to get good RPKM measurements from a cow. How many reads do you have?

Should i use DNA ref or RNA ref. for mapping using gs reference mapper ??

What data base set should be used for finding expression RPKM, from transcriptome reads of cow?

Sequencing was carried by 454 gs-flx.

[abpatel2288]

Thanks dear
jmartin for your reply, i having around 529087 reads with minimum length of 40 after removing MID and other adapters..

as you told RPKM values are average. i have used CLC gemone work bench and NextGene software's for RPKM.

Results of both softwares are having much difference in RPKM values of same reads, i am totally puzzled why i am getting so much difference in value, and on which i should rely for further analysis .

[jmartin127]

I see what you mean, I cannot say for sure why the values are so different from the two software programs. Are you aligning to the genome or to the transcripts themselves to calculate RPKM? Usually I work with Illumina reads, so the tools I typically use wouldn't be applicable to you unfortunately.

i have used CLC gemone work bench and NextGene software's for RPKM. Results of both softwares are having much difference in RPKM values of same reads, i am totally puzzled why i am getting so much difference in value, and on which i should rely for further analysis .

[abpatel2288]

I am aligning reads to genome... Is there any other option which i would check ?

[jmartin127]

Often when I've done this type of analysis, I've aligned my reads to the spliced transcript sequences. For most organisms this will be listed as the "XXX_cDNA.fasta", for example. To get the expression values you can basically count the number of reads aligning to each transcript, and then divide by the gene length and the number of reads in your sample to get RPKM (you would also have to multiply by 1000*1000000, since it is Reads Per Kilobase of exon model per Million mapped reads).

I am aligning reads to genome... Is there any other option which i would check ?

[vineeth_s]

Another way

If you have done RNA-Seq (which is what you have I would guess, considering you say transcriptome analysis), perhaps you could give Trinity, from the Broad Institute, a run

Trinity is a de novo transcriptome assembler, voiding the need for you to have a reference

And here's the paper

If you do use it, it would be great to hear what your experiences with it were

[abpatel2288]

will it work with 454, GS FLX