mRNA and miRNA sequencing

[amrita308]

Is it possible to prepare a single library for integrated mRNA and miRNA sequencing?

[nucacidhunter]

It is possible to develop such a method but I do not think any commercial kit is offered for this type of library prep. However, nanoString "nCounter miRGE Assay" allows simultaneous counting of miRNA and mRNA from total RNA input.

[NGSsequencer]

Currently there are no sequencing based methods that allow you to use the same sample to simultaneously sequence both large and small RNA. Remember, different transcript sizes, which could yield significantly different library sizes would also pose issues with sequencing. Clustering bias could be a real issue. Unless you have limited samples, split the isolate, and sequence in different lanes(if you have a HiSeq)

[amrita308]

Thanks alot.. It was very helpful..

[amrita308]

I have gone through the nanoString "nCounter miRGE Assay" but this gives information about the expression studies whereas I am searching for integrated mRNA and miRNA seq protocol. Could you please tell if you have done such integrated sequencing, also it would be a great help if you have come across any such related articles.

[nucacidhunter]

If there is a rRNA deplation reagent for your species then it is easy. Deplete rRNA and adjust washing steps to keep small RNA in. Then fragment the RNA and treat with PNK to phosphorylate the 5' ends. Small RNA-Seq library prep method or kit can be used for library prep from this material. You will need to skip size selection step after PCR which is performed for Small RNA library.

[amrita308]

i am curious whether if it can be done in this way : First doing rRNA depletion and then fragmenting the whole mRNA and miRNA's into same size , followed by end repairing, adapter ligation and size selection. Any suggestion?

[nucacidhunter]

An RT reaction and second strand synthesis need to be done prior to end repair. If you prime RT with randomers then most of the miRNA and small RNA will be lost.

[NGSsequencer]

i am curious whether if it can be done in this way : First doing rRNA depletion and then fragmenting the whole mRNA and miRNA's into same size , followed by end repairing, adapter ligation and size selection. Any suggestion?

IM just curious why you would want to do this? obviously unless this is a single cell.

miRNA size is 21bp, but the miRNA library size is 140-170nt+ depending on size. These libraries consist of 3'/5' adapters for size elongation for sequencer compatibility. plus the platform specific adapters. The presence of small libraries compared to library sizes for transcriptome would be different. Plus what read length are you reading? small RNA is 50-75bp, large RNA 150+

Im not saying you CANNOT do it, but the data itself will be challenged.

Garbage in, garbage out....it would literally be cheaper per usable mapped, de-duplicated, read to separate and sequence.