mRNA seq, very low clusters

[cub103]

Hi-,

I recently run an mRNA sequence using Illumina GAII following the instructions in the product manuals for all steps. It turned out that the clusters we got was very low (65K~180K) and so was the sequence reads. I am trying to figure out what might be the reasons that have caused this. So I am turning to your expertise and suggestions...Here are some of the information for our sequence:

• Total RNA from frozen human tissue (3-6 ug used)
• RIN for RNA are 5.3-7.3
• Library size 230-260bp quantified using Bioanalyzer 2100
• Illumina mRNA-Seq Prep Kit
• Illumina 36 Cycle Sequencing Kit v4
• Illumina single-read cluster generation kit v4

Thank you.

cub

[mnkyboy]

How much library did you load for cluster generation and how did you quantify it?

Is the sequence quality any good? If the quality is good you just did not load enough library.

[cub103]

The library was quantified using Agilent Bioanalyzer with DNA 1000 and we loaded 8 pM as specified by the protocol.

The sequence quality looks pretty good.

So if the loaded libraray was not engouh, how do I estimate how much more do I need to load for the repeat run?

Thank you.

[mnkyboy]

You should try to check the quantity by qPCR if you can or in the future the Bioanalyzer can lie about pM.

You now know however how much what you that was 8pM gives you in terms of cluster density so you can just multiply till you get the desired amount.

[cub103]

PCR primers

Thanks very much for the suggestion.

Here I founda set of PCR primers for quantifing my cDNA library using qPCR, do not know if they the right ones or not?

S: ATG ATA CGG CGA CCA CCG AG
A: CAA GCA GAA GAC GGC ATA CGA G

Thanks.

[mnkyboy]

Most people follow the attached Illumina protocol or use the Kapa kits. The Kapa kits work very very well for mRNA-seq libraries. There might be a newer version of the Illumina protocol I just stick with the Kapa kits.

[cub103]

Great! Thanks a lot!